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Identification of traits and QTLs contributing to salt tolerance in rice (Oryza sativa L.) Thieu Thi Phong Thu 2019
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Identification of traits and QTLs contributing to salt tolerance in rice (Oryza sativa L.) A dissertation submitted to Graduate School of Bioscience and Bioenvironmental Science, Faculty of Agriculture, Kyushu University in partial fulfillment of the requirement for the degree of Doctor of Philosophy by Thieu Thi Phong Thu Laboratory of Plant Nutrition Japan, 2019
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Table of contents Acknowledgment i Abbreviation ii Summary iii-vi Chapter 1 General introduction 1-10 Chapter 2 Effects of salt stress on plant growth characteristics and 11-34 mineral contents in diverse rice genotypes Chapter 3 Allocation of macronutrients in roots, sheaths, and leaves 35-56 determines salt tolerance in rice Chapter 4 Determination of sampling method to identify salt tolerant 57-75 traits in rice Chapter 5 Identification of QTLs by genome wide association study 76-110 (GWAS) in rice for salt tolerance Chapter 6 General discussion 111-122 Appendix 123-129 References 130-146
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Acknowledgment I would like to express my sincerest appreciation and deepest gratitude to the following organizations and individuals in the pursuit of my Doctoral degree: Vietnam Government, Ministry of Agriculture and Rural Development, and Ministry of Education and Training for granting me the scholarship; Vietnam National University of Agriculture, and Department of Cultivation Science for allowing me to leave to go on studyand their kind supports; My supervisor Assoc. Prof. Dr. Takeo Yamakawa for his guidance, encouragement with all his heart and invaluable advices throughout my research; Prof. Dr. Ken Matsuoka, Assoc. Prof. Dr. Akiko Nakashita-Maruyama, Dr. Masamichi Kikuchi from Laboratory of Plant Nutrition; and Prof. Dr. Hideshi Yasui, Dr. Yoshiyuki Yamagata from Laboratory of Plant Breeding for their kind helps and invaluable comments and suggestions during my study; All members of Plant Nutrition Laboratory for their kind understanding, their enthusiasm supports and helps during my time in Japan; All my Vietnamese friends and other International friends for their helps and moral supports which made my stay in Japan a pleasant and memorable one; Vietnam international education development (VIED) at Ministry of Education and Training and Mrs. Nguyen Thi Thanh Tam, an officer of Students Affairs Division, for their kind understanding, their enthusiasm supports and help me to complete both the necessary proceeds in Viet Nam and Japan. Last, but not least to my husband Duong Thanh Huan, my little daughter Duong Hong Ngoc and my parents, my parent’s in-law, my sisters and their family, my husband’s older brother and his family for their love and moral support. i
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Abbreviation 3LM = Three leaves method ASW = Artificial seawater C2S = Cutting part of leaf sheath between base and up to 2 cm Ca = Calcium CAS = Cutting part of leaf sheath between 2 cm from base to collar Chr = Chromosome CM = Cutting method CT = Control treatment DW = Dry weight EC = Electrical conductivity GWAS = Genome wide association study HDRA = High density rice array HS = High salt susceptible group HSD = Honesty significant difference IB = Indica Block JB = Japonica Block K = Potassium KCR = Kyushu university cultivated rice L = Leaf LD = Linkage disequilibrium LW_R = Leaf dried weight reduction Mg = Magnesium MT = Moderately salt tolerant group N = Nitrogen Na = Sodium P = Phosphorous PCI = Pearson correlation index QTLs = Quantitative trait loci S = Salt susceptible group Sh = Sheath ShtL_R = Shoot length reduction ShtW_R = Sheath dried weight reduction SES = Standard evaluation score SNP = Single nucleotide polymorphisms SOS = Salt overly sensitive ST = Salt stress treatment ST/CT = Salt stress treatment/Control treatment T = Salt tolerant group WRC = World rice collection Y-sol = Yoshida solution ii
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Thesis Summary The global population is expected to reach to 9 billion people by 2050, increasing the pressure for agricultural production in marginal saline lands. Agriculture will have to increase its crop productivity by 70-110% in 2050 to feed that world. Globally, it is estimated that 45 million of the 230 million ha of irrigated lands (19.5%) have to suffer from salinization problems by various degrees. This is a serious problem because irrigated areas are responsible for one-third of world food production. Therefore, agriculture production has been faced to not only increase crop yields that have not been seen before but also have to do this in a changing climate. Development of the salt tolerant varieties has been considered one of the key strategies to increase rice production in the coastal area through breeding by genetic methods. The important steps have been pointed out for genetic method of improving salt tolerance in rice such as evaluation of variation of genetic sources for salt tolerance in rice, identification of molecular markers associated with QTLs or genes conferred tolerance to salt stress, discovery of genes regulating salt tolerance and development of cultivars harboring those salt tolerance genes. Salinity tolerance in rice is regulated by multiple genes. Expression of salinity tolerance is the result of many physiological and biochemical activities in plants. Therefore, determining the representative traits for salinity tolerance is a very difficult but extremely important task. The objectives of this research are to determine the optimal salinity tolerant traits, the effective sampling method, and finally to find QTLs contributing to improving salt tolerance in rice. Thirty-seven rice varieties were screened for salt tolerance using a hydroponic system with ion components similar to seawater in growth chamber. Two-week-old seedlings were grown for 7 days on Yoshida hydroponic solution. The treatment group then additionally received an artificial seawater solution (electrical conductivity, 12 dS m−1). After a 2-week period of salt stress, standard evaluation scores (SES) of visual salt injuries iii
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were assessed. The K, Na, Mg, and Ca contents were then determined for the roots, sheaths, and leaves. Following the SES results, the 37 varieties were divided into four groups: salt- tolerant (STGs), moderately salt-tolerant (MSTGs), salt-sensitive (SSGs), and highly salt- sensitive (HSSGs). In the control treatment, STGs had the highest sheath K content, whereas HSSGs had the lowest. Sheath Kcontent was also highly and negatively correlated with SES. This suggests that sheath K may be useful for identifying salt-tolerant varieties under non- saline conditions. Plant growth was significantly affected under salt stress, but STGs had the smallest decrease in dry weight of sheath. SES was significantly correlated with sheath and leaf Na, sheath K and Mg contents, and sheath and leaf Na/K and Na/Mg ratios. The results suggested that sheath K content, Na/K, and Na/Mg ratios may be useful indicators for genetic analyses of salt-tolerant varieties under salt stress conditions. Another set of twenty-nine rice varieties were screened for salt tolerance using a hydroponic system with ion components similar to seawater in a green house. Following the SES results, the 29 varieties were divided into three groups: salt tolerant (STG), moderately salt tolerant (MSTG), salt susceptible (SSG). Under the control treatment, salt tolerant varieties exhibited low K content in root. Under the salt stress treatment, the salt tolerant varieties exhibited low SES, high N content in leaves and sheaths, low Na content in leaves and sheaths, low Mg contents in leaves and sheaths, and low Ca content in sheaths. The salt tolerant varieties also showed high ratio of sheath dry matter in salt stress treatment and sheath dry matter in control treatment, high N content in leaves and sheaths, high K content in sheaths, and low Na/K ratios in leaves and sheaths. Therefore, these parameters might be useful to understand salt tolerance in rice. Four rice varieties were used in sampling method experiment in phytotron. In the control, Yoshida solution was used continuously during the experiment. In the salt treatment, seedlings were grown for two weeks in a 12 dS m−1 salt solution. Two sampling methods iv
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were tested including cutting method and three leaves method. The results showed that cutting method and three leaves method are useful for experiments related to salt tolerance in rice. The mineral contents in salt tolerant cultivars was significantly different in comparison with salt susceptible variety in cutting part of leaf sheath between 2 cm and collar (CAS) and leaf blade. The CAS and the leaf blade were useful for identifying salt tolerant related traits. Leaving roots and shoots from the base to 2 cm might be possible to continue the growth of seedling and produce seeds which will be used for re-phenotyping of the progeny. The first three leaves may not be needed for sampling to determine mineral contents. As a result, it is possible to take a sample without the first three leaves and using cutting part of leaf sheath between 2 cm and collar. The K content, Na content, Mg content and Na/K ratio in CAS and Na content, Na/K ratio in leaf blade should be used as salt tolerant traits in molecular and genetic analysis. A genome wide association study (GWAS) was performed to identify potential QTLs associated with salt stress tolerance in rice by correlating the genotyping data set with the phenotypic expression of 225 diverse rice accessions for 11 biochemical and agronomic traits. GWAS was run using a mixed linear model (MLM) and population parameters previously defined (P3D) in Tassel 5.0 and predicts genomic regions associated with traits for Japonica subpopulation and Indica subpopulation. To analyze single nucleotide polymorphisms (SNP) calls, a high-density rice array of 700,000 SNPs was used, which derived from the sequencing of wild and domesticated rice. The kinship matrix (K) estimated from SNP genotyping data. Association signals at the same QTL block were confirmed location on chromosome by analysis of linkage disequilibrium (LD) block using gPlink- 2.050 and Halotypeview version 4.2. GWAS resulted in detection of numerous SNP markers scattered over the rice genome that were associated with various salt tolerance traits. A QTLs region on chromosome 3 was found to contribute to the variation in salt tolerance in Indica v
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subpopulation and related to two traits of sheath Ca content and sheath Mg content. Three QTLs regions on chromosome 2, 4, 5 were found to contribute to the variation in salt tolerance in Japonica subpopulation and related various traits of sheath Na content, sheath Mg content, sheath Na/K ratio, leaf Na content, leaf Na/k ratio. GWAS and LD analysis showed several SNP makers highly related to salt tolerant traits. The results of this thesis dressed out both the insight in traits and mechanisms related to salt tolerance and sampling method for salt stress experiment in rice. The underlying genetics as presented in this thesis is of direct use to breeders and scientists and will significantly contribute to improving the salt tolerance traits in rice. vi
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Chapter 1 General introduction 1.1. The salinization and current worldwide problems The global population is expected to reach to 9 billion people by 2050, increasing the pressure for agricultural production in marginal saline lands (Negrão et al. 2011). Global food production will need to increase by approximately 50% by 2050 to match population growth (Flowers 2004; Rengasamy 2006). Tester and Langridge (2010) also reported that crop productivity of agriculture has to increase by 70-110% in 2050 to feed that world. However, agricultural production faces increasing challenges from salt intrusion due to global warming, ice melt, and rising sea levels. The area and salinity level of salt-affected soil have been increasing in parallel with the increase of world population. Globally, it is estimated that saline and sodic soils cover 397 million and 434 million ha (FAO 2016), respectively, while 45 million ha of irrigated lands (19.5%) have been affected by salinization. This is a serious problem because irrigated areas are responsible for one-third of world food production (Linh et al. 2012). In addition, many factors that related with climate change like water scarcity, elevated temperature, flooding and salinity cause abiotic stress and bad effect on crop productivity. Therefore, agriculture production has been faced to not only increase crop yields to a level that have not been seen before but also have to do this in a changing climate (Roy et al. 2011). Among abiotic stresses, salt stress is noticed as the most serious factor (Tuteja 2007). High salinity have predicted that will be damaged to 20% of total cultivated area and 33% of irrigated land. And, the salinized areas are growing to 10% a year because of various problems, including low precipitation, high surface evaporation, using salty water for irrigation, and poor agricultural practices. Until 2050, it is considered that more than half of 1
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the arable land would be salinized (Jamil et al., 2011). Soil salinization widespread in humid regions such as South and Southeast Asia (Szabolcs and Pessarakli, 2010), where rice is a staple food crop (Kobayashi et al. 2017). Many high-output rice cultivation regions have been faced with salinization because these regions are located in coastal areas. Therefore, it might cause a lot of bad impact on the productivity of rice. Salt tolerant cultivars may provide opportunities to improve the salinity tolerance of rice through breeding. One of the main strategies that has been focused is producing the salt tolerant varieties in saline region through breeding by genetic methods to increase rice production. The important steps have been pointed out for genetic method of improving salt tolerance in rice such as investigation of genetic sources variation for salt tolerance, determination of molecular markers related with QTLs or genes regulated tolerance to saline condition, identification of genes regulating salt tolerance and introduce those salt-tolerance genes to elite cultivars (Sahi et al. 2006). 1.2. The saline soil and adverse effects of salt stress on plant The saline soil The saline soil refers to a soil that contains sufficient soluble salts to impair its productivity. The soil soluble salts include various ratios of the cations (Na+, Ca2+ and Mg2+), and the anions (Cl-, SO 2-). The most popular ions that related to soil salinity are Ca2+, Mg2+, 4 and Na+ (Alam 1999). Saline soils are identified if its EC is 4 dS m-1 or more (about 40 mM NaCl) (Munns and Tester 2008). The condition is assumed as salinity when its EC cause the decrease of most crop. The adverse effects of salt stress on plant Salinity severely limited the crop production because of inducing an abiotic stress to 2
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plant (Shannon 1998). In general, two types of stresses on plant come from salinity are osmotic and ionic stresses. Osmotic stress happens when the salt concentration around the roots increase over the threshold of plant tolerance. Ionic stress occurs in case of increase of Na+ content in old leaves lead to the influx of large amounts of Na+ inside plant body. It results in high Na+ content in the vacuole and cytoplasm, then cause bad effect on metabolic processes and cell death (Munns and Teaser 2008). There are three ways which salt stress affects on the plant growth and development. The first is by reducing the soil osmotic potential and inducing the bad situation that water can not enter the roots. The second is ion toxicity resulting into changes of Na+/K+ ratios. The third is interruption or preventing the absorption of other mineral elements. As a consequence, these ways results the poor growth and plant death (Mc Cure and Hanson 1990). Salt stress can induce the physiological drought problems as well as the ion toxicity (Zhu 2001). Thus, stresses of salt and drought has bad effects on many processes of plant physiology and metabolism and be reasons of stresses such as hyper ionic and hyper osmotic that severely depress the plant growth. Moreover, salt stress also makes the changes in K/Na ratio because of ion-specific stresses. The external Na+ might cause the negative effect on the intracellular K+ influx. And, salt stress increases the cytosol Na+ and Cl- concentrations and damage on the cell. The membrane potential can be broken by high Na+ concentration, then a good condition for Cl- absorption is established. In addition, level of Na+ content of above 100 mM damage to cell metabolism and limit activities of enzymes, cell division and expansion, and membrane structure. This also decreases the photosynthesis and produces reactive oxygen species. K ion is a crucial element necessary for plant growth. Changes in K+ concentration can interrupt the osmotic balance, the stomata and enzyme functions (Singh and Sengar 2014). The growth up of Na+ concentration because of salt stress induces destructive 3
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consequences such as the retarded plant growth (Saqib et al. 2008), slowing down flowering and poor fertility, bad development of panicle and grain in rice (Zeng and Shannon 2000, Abdullah et al. 2001, Kato et al. 2008, Rao et al. 2008), reduced K+ and Ca2+ concentrations (Fageria et al. 2012), inhibition of photosynthetic activity (Netondo et al 2004, Chaum et al. 2006, Chaves et al. 2009, Moradi and Ismail 2007). 1.3. The salt tolerance mechanism in rice Halophytes and glycophytes are two groups of salt tolerant plant and salt susceptible plant. The halophyte plants can tolerate to high saline condition of 400 mM NaCl, while glycophytes can not (Mass and Nieman 1978). Among agriculture crops, few crops are halophytes, and large proportions of crops are sensitive to salinity stress. With in cereals, the highest tolerance to salt stress specie is rye (Secale cereale) with a stress threshold of 11 dS m−1 EC. While, the lowest tolerance crop is rice with a salt stress threshold of 3 dS m−1 EC (USDA, 2016). The reduction of yields can reach to 50% once rice is cultivated under 6 dS m-1 EC salinity level (Maas 1990, Maas and Hoffman 1977, Ren 2005). The rice growth periods of germination, active tillering and towards maturity are tolerant to salt stress. The seedling, flowering and early reproductive stages are considered as be susceptible to saline condition. (Singh et al. 2008).To deal with salty problems, various mechanisms were seen in rice including biosynthesis and accumulation of osmolytes for osmoprotection, ion homeostasis and compartmentation, antioxidants, reactive oxygen species (ROS) detoxification, and programmed cell death (Hoang et al. 2016). Munns and Tester 2008 reported that ion exclusion and osmotic tolerance are main ways which happen in salt tolerant rice varieties. These ways are also assumed ion exclusion, osmotic tolerance and tissue tolerance (Roy et al. 2014). To reduce the over leaf accumulation of Na+ and Cl–, ion exclusion mainly involves Na+ and Cl– transport processes 4
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in roots. Ion exclusion includes two pathways, one is the retrieval of Na+ from xylem, and the other occurred in root system by returning of ions back to the soil. Osmotic tolerance is happened before shoot Na+ accumulation, this is controlled by long distance signals and relates to drought stress which caused by salt stress to protect leaf expansion and stomatal conductance (Rajendran et al. 2009). Tissue tolerance is related to vacuole accumulation of sodium, synthesis of compatible solutes and production of catalyzing detoxification of ROS enzymes. Intracellular cation compartmentation To deal with salt stress, one of the mechanisms is to reduce roots Na+ uptake and keep low leaf content of Na+. Munns and Tester 2008 reported that if Na+ exclusion is not preformed well, toxic effects occurred some days or weeks after, then older leaves start dying. In both Indica and Japonica varieties, correlation of leaf Na+ content and salt tolerant degree was found (Moradi and Ismail 2007). Rice young leaf is protected from salt stress because old leaf accumulated toxic ions. Wang et al. (2012) showed that under salt stress condition, old leaves have higher contents of Na+, Cl– and NO – compared with young leaves. Under 3 salinity, up-regulation of OsHKT1;1, OsHAK10 and OsHAK16 promotes old leaves accumulate Na+ ion and increased OsNHX1 expression improves the distribution of Na+ into old leaves. Rice panicle has lower salt content in saline tolerant cultivars, and compared to husks and rachis, grains have lowest salt contents (IRRI 2018). The healthy level of flag leaf is an important parameter for good yield under salt stress condition. Evaluation of mineral content is flag leaf shows lower salt content in this leaf in salt tolerant cultivars (Ahmad et al. 2013). Thus, flag leaf should be selected for evaluating tolerance to salt stress at the reproductive stage (IRRI 2018). 5
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Under the salt stress condition, salt tolerant varieties showed lower Na+ in leaves and shoots in comparison with salt susceptible varieties (Dionisio-Sese and Tobita 2000, Lee et al. 2003, Moradi et al. 2007, Ghosh et al. 2011). Pokkali, a saline tolerant variety, showed low accumulation of Na in cytosol because of decrease the Na uptake, temporary accumulating in the cytoplasm, and quickly extruding into vacuoles (Kader and Lindberg 2005). The sodium ion exclusion and the regulation of sodium transporter was studied in many researches. It is because a toxic level of Na+ happens before Cl– ion reaches to damage threshold (Munns and Tester 2008). Lin and Kao (2001) also concentrated on Na+ instead of Cl– because negative correlations between shoots and roots Na+ contents and salt stress tolerance were found. Cell ion homeostasis and cation transporter One of the important parameters that shows a cultivar is tolerant to salt stress is keeping a low cytosolic Na+/K+ ratio. This is necessary to have an optimal ionic homeostasis in plant cells under salt stress condition (Maathuis and Amtmann 1999, Cuin et al. 2003), and support to photosynthesis and plant growth (Rodrigues et al. 2013). Maintaining low cytosolic Na+/K+ ratio may be because of maintaining low Na+ content or high K+ content or both phenomenon. Under salt stress condition, rice varieties with different salt tolerance showed the mineral contents were different in roots, leaf sheaths and leaf blades. Salt tolerant varieties showed higher K content, lower Na content, lower Mg content and lower Na/K ratio in leaf sheath and lower Na content, lower Na/K ratio in leaf blades than salt susceptible varieties. (Thu et al. 2017, Thu et al. 2018). Relating to a high ratio of K+/Na+, identification and characterization of genes and transporters have been done. Wang et al. (2012) reported that under saline condition, up- 6
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