Salinity is mainly a problem of irrigated systems in arid and semi-arid regions with insufficient precipitation, unlike drought, which mostly affects rainfed systems, has the highest reduction in yield potential. Although, saline soils are result of natural, geological, hydrological and pedological processes, there are other factors like deforestation, contamination with chemicals and overgrazing contribute towards soil salinity. Yield loss from osmotic stress in plants mainly due to high salt accumulation in soil water causes salinity is an important abiotic stress over the globe. Decreased osmotic potential in soil leads to deterioration of soil physical properties like permeability and aeration, which finally reduces the availability of fresh ground water. To mitigate these problems of saline soil, resistant plants against salt stress is a way to overcome the yield loss. There are many resistant sources available naturally, mostly in wild relatives and local landraces of crops, which are yet to improve for other desirable qualities. Isolation of the genetic region associated with the desired trait from those sources and introgression in improved lines is an approach to develop more resistant varieties of the crop. IR 107321-1-141-3-120, a highly resistant pyramidal rice line and IRRI 154, a susceptible rice line for salt stress are used in current research. In this project, previously defined three QTLs, namely qSES1L, qSES1S and qSES11S from the cross between donor line 141 and recipient line 154 are evaluated by prediction of effectiveness of the QTLs under different backgrounds and candidate genes will be identified along with their expression level via fine mapping and RNA-seq technology. The resulted reduced genetic region or gene/genes associated with the salt resistance can be further used in introgression program.
Intellectual merit
This research project focuses in understanding the genetic mechanisms of salinity tolerance through study of the effectiveness of the qtls under different elite backgrounds. Fine-mapping of NIL and BIL families to refinement of the candidate gene and transcriptome profiles of tolerant and sensitive lines will be examined with and without salt stress via RNA-sequencing. Gene expression analysis through RNA-seq is to discover, quantify and profile RNA. This will be combined with available QTL and comparative genomics results to inform a candidate gene analysis.
Broader impacts
Abiotic stresses like salinity and drought account for 20-50% yield loss in the most important food crops. Rice is considered one of the most salt-sensitive crop species, in which high salt in soil can cause measurable yield declines. With the increasing population with other environmental problems like pollution, scarcity of fresh water in different regions of the world, use of drainage or irrigation water with salt content is increasing. Developing resistant variety against salt stress is the most favorable approach to mitigate the problem of saline soil and its detrimental effects in crops. For this purpose, I am working in International Rice Research Institute, Phillipines, collaborating with University of Illinois, Urbana Champaign to find novel gene/genes responsible for salt resistant in rice.
Introduction
Saline soils are result of natural, geological, hydrological and pedological processes, there are other factors like deforestation, contamination with chemicals and overgrazing contribute towards soil salinity. Natural and human activities induce the accumulation of salts in soil water. Salinity causes a secondary effect in soils, termed as sodicity, which occurs leaching of soluble salts into the sub soil and sodium binds with negative charge. Irrigated land causes secondary salinization due to poor leaching capacity of the soil with insufficient rainfall, which inhibits the absorption of essential nutrients from the soil. More than 6% of the world’s land is contaminated with salts, stated by FAO Land and Nutrition Management service, 2008 (Yadav et al., 2011). Moreover, 10% increase of saline areas per annum has been recorded due to various regions like low rainfall, high surface evaporation, weathering of native rocks, irrigation with saline water and poor agricultural practice. By 2050, 50% of the arable land will be salinized (Jamil et al., 2011). Salinization has a big impact on human health through environmental hazards, which affects more than 1 billion globally (Metternicht and Zinck, 2003; Yensin, 2008). 7million hectors of land in India is under effect of salt stress (Patel et al., 2011). Along with low agricultural production of crops, salinity has a bad hazardous impact on physiochemical properties of the soil and ecological balance mainly due to soil erosion. Saline soils hamper the growth of the plant (Paul, 2012). Although, there are many QTL regions for salt stress has been reported over years, which involves more genes with different salt tolerance mechanism, finding the exact locus of these QTLs are needed a lot of work and attention to identify novel donors and introgression in marker assisted selection. Identification of the genes responsible for salt tolerant is the key way to improve the susceptible plants. For this purpose, the genetic basis of the mechanisms against high salt effect in plant is to be evaluated. Tolerance of rice to accumulated salt in the soil can be a way to reduce the effects of this abiotic stress. Many QTLs associated with salt tolerant have been mapped, but none are in widespread use in breeding activities.
Background
Salinity of a soil can be measured by the electrical conductivity of the saturation extract (ECc). A soil is considered as saline soil, if the electrical conductivity of the saturation extract in the root zone of a plant surpasses 4 dSm-1, which is around 40 mM of NaCl at 250 C (Shrivastava et al., 2015). Although most of the crops show poor yield at this ECs, many other crops are recognized as poor yielded at lower ECs than this (Munns, 2005: Jamil et al., 2011). Saline soils are categorized by low (0.25 dS m-1), medium (0.25 to 0.75 dS m-1) and high (0.75 to 2.25 dS m-1) on the basis of electrical conductivity (US Salinity Laboratory Staff, 1954). Crops response in a wide range, while exposed in salinity stress. The extreme concentrations of these ions can make scarcity of available water for crops by decreasing osmotic potential of soil along with deterioration of soil physical structure. Ionic and water imbalance in soil and crop by diminishing the water permeability and soil aeration leads to limited growth in crops and poor yield as it alters the K+/ Na+ ratio with increasing amount of Na+ and Cl-. Alteration of ionic homeostasis leads to more uptake of Na+ through K+ transport channels to the cell, which effects in enzymatic activities. Apart from the reduction of yield in crops, salinity has a major impact in physiological, chemical and ecological properties of soil. Soil erosion is the major outcome of sodic soil along with deleterious effects on germination, vegetative and reproductive development in crops. Ground water contamination is caused by accumulation of different cations and anions, for example Na+, Ca2+, Mg2+, Cl-, SO42-, HCO3-. Cl- and Na+ are considered as most toxic ions in soils, as they have deleterious effects in crops. In a nutshell, extreme accumulation of salts in soil causes ion toxicity, osmotic stress, nutrient deficiency and oxidative stress results in alteration of physiological, biochemical and molecular characteristics in plants (Munns and James, 2003, Tester and Davenport, 2003).
Plants have a wide range of mechanisms to overcome the salt stress. The ability of a plant to adapt in high salt concentrations is defined as resistance, which is the combination of tolerance and avoidance (Levit et al., 1980). Plants can avoid high salt stress by delayed germination and maturity until the favorable conditions prevail, high salt elimination from the plant and deposition in roots and distribution of salts in various salt glands along with older leaves (Hasegawa et al., 1986). Removal of sodium from cytoplasm and store in vacuoles is another mechanism of salt resistance triggered by salt inducible enzyme Na+/H+ antiporter (Apse et al., 1999). Osmotic adjustment can be facilitated by compartmentalization of the salt ions in a plant under salt stress is an important way of salt tolerance (Guerrior, 1996). In stress condition higher amount of salts can make accumulation of other compatible solutes like protein, glycine betaine (Hasegawa et al., 2000; Zhifang and Loescher, 2003; Ghoulalm et al., 2002; Giriza et al., 2002; Khan et al., 2000; Wang and Nii, 2000), which function as osmotic adjustment, protection of enzymes, membranes and storage of house of energy and nitrogen (Bandurska, 1993; Perez- Alfacea et al., 1993). Delayed germination is one example of avoidance mechanism of plant under salt stress. Some other physiological and biochemical mechanism of salt tolerance are ion homeostasis and compartmentalization, ion transport and uptake, biosynthesis of osmoprotectants, activation of antioxidant enzyme and synthesis of antioxidant compounds, synthesis of polyamines, generation of nitric oxide and hormone modulation. Transporters in rice plant take salt first to older leaves than younger leaves and lastly inflorescence. Plants transport salt to next older leaves before the salt accumulated old leaves died (yeo and Flowers, 1982).
Genetics of salt tolerance in rice has started with searching for the QTLs and genes responsible for salinity tolerance in different germplasms. Various genetic regions were identified in rice for salt tolerance, among which “Saltol” is the major QTL in rice chromosome 1, derived from the variety Pokkali (Bonilla et al., 2002). In addition, “Saltol” was reported as major QTL for salinity tolerance, named as SKC1 or (OSHKT1; 5), reported in Nana Bokra (Lin et al., 2004). Na/K homeostasis is maintained by “Saltol” locus under salinity stress was reported (Lin et al., 2004; Ren et al., 2005; Thomson et al., 2010; Platten et al., 2013).
Different approaches like diversity analysis, genetic mapping, and association analysis prefer SNP marker system to find new alleles for its robust and high polymorphic characters. (Rafalski, 2002; Chagne et al., 2007; Ganal et al., 2009, Thomson, 2014). 384-SNP assay in different sub clusters of rice genus to characterize allelic diversity at “saltol” locus specified novel donors for salt tolerance (Rahman et al., 2016). In rice shoot and root, 8 qtls related with different physiological traits were identified in 5 chromosomal regions for salt tolerance (Lin et al., 2004). Among those 8 qtls, qSNC-7 and qSKC-1 has major phenotypic variance for Na+ concentration and K+ concentration in shoot respectively. It was reported that, these qtls define different positions in genome; therefore, genes responsible for Na+ and K+ accumulation in shoot are different. Marker locus RG13 was reported on chromosome 5 for salt tolerance which shows 11.6% of total phenotypic variance in seedling survival under salt stress (Lin et al.,1998). RILs developed from a cross between salt susceptible IR29 and tolerant Hasawi facilitated 20 novel QTLs in chromosomes 1, 2, 4, 6, 8, 9 of rice (Bizimana et al., 2017). SKC1 qtl includes gene of transporter group like HKT type in parenchyma cells of xylem vessels function as Na+ transporter, which maintains K+/Na+ homeostasis in plants under salt stress (Ren et al., 2005). From a cross of a susceptible line Koshihikari and Nona bokra, 8 qtls correlated with K+ and Na+ content was mapped in rice plants in salt stress (Ren et al., 2005). SKC1 is the major QTL found after analyzing 2973 BC3F2 plants of a high-resolution map. Abundance of mRNA for SKC1 was identified in rice root than shoot carried out with RT PCR analysis in NIL population (Ren et al., 2005). This is supported by expression of TaHKT1 in wheat (Schachtman et al., 1994) and AtHKT1 in A. Thaliana (Uozumi et al., 2000). Upregulation of SKC1 transcripts under salt stress in roots but not in shoots reveals about expression patterns of ATHKT1 genes. SKC1 regulates K+ and Na+ translocation between shoot and root, K+/Na+ homeostasis in shoot.
Transcription factor genes were identified among the differentially expressed genes, revealed about involvement of different regulatory pathways of salt stress tolerance through transcriptional regulation of downstream genes. Upregulation of one transcription factor “bzip” in salt sensitive wheat upon long term exposure of salt stress had been seen (Johnson et al., 2002). Other transcriptional factors like OsNAC5 and ZFP179 were increased in salinity tolerant crops, which lead to accession of proline, sugar and LEA proteins under salt stress having some mechanisms for resistance of saline soil (Song et al., 2011). In saline conditions, upregulation of some genes and transcriptions factors has been recorded in various plant species, which are responsible for ion transport, and homeostasis, senescence function as molecular chaperones with ROS- scavenging and osmotic regulation. Increased glutathione S transferase and ascorbate peroxidase in saline condition has been reported, which are responsible for ROS-scavenging (Kawasaki et al., 2001). Some genes are downregulated upon exposure of salt stress which enhances some other factors in plants to provide salinity tolerance. Down regulation of B- carotene and total carotenoids helps potato to overcome salt stress (kim et al., 2012). Both upregulated and downregulated, differentially expressed transcripts were reported in leaves and roots of Dongxiang wild rice (oryza rufipogon Griff.) after exposing of salt stress (Yi Zhou et al., 2016).
Previously, some novel QTLs were identified for seedling stage salinity tolerance in rice by using a mapping population of a cross between susceptible line IRRI 154 and highly tolerant pyramidal line IR 107321-1-141-3-120. Phenotyping for the salt tolerant was carried out in seedling stage of the population by using SES score or visual injury score. Three QTLs were identified on chromosome 1, which has LOD score >4 and one QTL with LOD >20 was found on chromosome 11. Among all the QTLS, qSES1.3 and qSES11.1 were correlated with visual injury score at all stages of phenotyping. This may lead to resistance for a long-term exposure of salt. On the contrary, qSES1.2 was found to be associated with early stage of injury, while, qSES1.1 on short arm of chromosome 1 is associated with later periods of salt injury. In previous work, candidate genes for all the QTLs have been identified. My research will focus on below mentioned objectives-
1. Assess effectiveness of QTLs in several improved genomic backgrounds.
2. Fine-mapping of QTL (qSES1.3/qSES1L) in the IRRI 154 background.
3. Evaluate the expression patterns of candidate genes for salt tolerance in rice by RNAseq technology.
Preliminary results:
Determination of the effectiveness of the QTL qSES11.1, qSES1.1 and qSES1.3 under different backgrounds
The highly tolerant IR107321-1-141-3-120 line (“141”) was crossed with different susceptible lines and different populations were developed through backcrossing followed by selfing. For This experiment, 100% genomic purity of the lines for the recipient background is not required for evaluating the targeted qtls under different elite backgrounds due to accessibility of the qtls in early generations. Various backcrossing and selfing generations for different genetic backgrounds were used to attain a range of genetic purity, which is lesser than 100%.Transcriptome is the overall transcripts present in a cell of an organism in a specific developmental stage. Molecular constitutes in a cell and their functions can be interpreted through study of the transcriptome in different growth and developmental stage of the organism. Evaluation of mRNAs, smallRNAs and non-coding RNAs in cells or tissues provides the information about the function of the specific genes along with posttranscriptional modifications and expression pattern of genes under different environments. Transcriptional analysis is an important approach to determine the upregulation or down regulation of RNA to screen candidate genes responsible for any biotic and abiotic stress tolerant. A group of gene regulators, named as transcription factors controlling the gene expression by binding in the cis acting elements of the promoter regions to target genes were identified. RNA-seq is one of the most important approach for transcription profiling of a genome providing precise and accurate expression levels and quantification of transcripts. Here, I propose the deep sequencing of cDNA library from the whole RNA of desired lines after exposure of salt stress under seedling stage to quantify the transcripts and its expression level for resistance of the stress.