GERMPLASM EVALUATION FOR SALT TOLERANCE IN JAPONICA RICE

Soil salinity is one of the environmental constraints that affect crop cultivation worldwide. Among cereals, rice (Oryza sativa L.) is one of the most salt-sensitive although cultivars can differ in their response to salt stress. In European coastal rice areas, the salt wedge intrusion phenomenon caused by the rise in the sea levels consequent to the ongoing climate changes is provoking a tendency toward salinization in the adjacent paddy fields and coastal areas where rice is grown. Thus, the identification of rice cultivars tolerant to salt stress and the dissection of salt stress tolerance mechanisms are of high interest for European rice breeding. Plant responses to salt stress are complex depending on the combination of the activity of many metabolic pathways and of the several involved genes, and thus they are difficult to control and/or engineer. Exploiting natural variation that occurs in worldwide genotypes may be a powerful approach to discover new genes and/or alleles involved in salt tolerance. Since salinity has many different effects on plants, several different tolerance mechanisms (osmotic tolerance, ion exclusion and/or tissue tolerance) exist. The predominance of a specific tolerance mechanism over others depends on salt stress intensity (severity x duration) and plant developmental stage. Concerning indica rice subspecies, several QTLs and some genes involved in the phenotypic variability in response to salt stress have been identified and exploited in breeding programs for genetic improvement of élite varieties. On the contrary, in the case of japonica subspecies, far fewer information are available. General objective of this work is the discovery of novel molecular variability at different developmental stages in japonica rice cultivars resulting in tolerance to the mild salt stress conditionsreported for some European rice areas. In major detail, possible research deliverables are the identification of QTLs, alleles and/or molecular markers exploitable in future genetic improvement programs. The stated goal was pursued by a Genome Wide Association Study (GWAS) using a panel of 277 accessions of japonica rice already genotyped-by-sequencing producing 31,421 SNPs. The accession panel was subjected to a two-year phenotyping through the evaluation, at different growth stages, of traits [leaf visual injuries score (SES), tillering rate, plant height, flowering time, flag leaf chlorophyll index, and flowering time] sensitive to salt stress. For these activities, plants were grown in greenhouse environment, in pots filled with paddy soil maintained submerged and, starting from the 4th leaf stage, subjected to about 5 dSm-1soil electric conductivity (ECe) by the addition of adequate NaCl amounts.For all the measurements obtained from the two-years data, the Least Mean Square (LMS) were calculated. To be able to compare two-year data and different parameters with different range amplitudes, all data were standardized by Z-score transformation, and subjected to two-step cluster analysis, achieving a Core Collection of the 5 most tolerant and the 5 most susceptible varieties in response to salt stress. Moreover, the seed germination dynamics and the seedling emergence rate were analyzed under high salt environment (150 mM NaCl for germination, and about 10 dSm-1 soil ECe for emergence) in a single-year experiment. For each of the 277 rice accessions the effect of salt on each trait was evaluated in terms of Stress Susceptibility Index (SSI) evaluating the performance of a single accession in relation to the whole collection. An association analysis between the phenotyping and the GBS results was carried out using Tassel 3.0 to calculate a Mixed Linear Model (MLM). The critical p-values for assessing the significance of SNPs were calculated based on a False Discovery Rate (FDR) separately for each trait; a FDR cut-off of α 0.05 was used for determining significance. Currently, a total of 33 Marker-Trait Associations (MTAs) between SNPs and the analyzed salt stress-related traits have been identified. Several loci were subsequently identified by intersecting the MTAs with the genes annotated on the Nipponbare reference genome. GWAS analysis carried out on germination and emergence parameters highlighted the presence of interesting associations between two markers and two loci in linkage disequilibrium with them: Os09g0369400 and Os07g0485000, coding for a Trehalose 6-Phosphate Phosphatase 7 (OsTPP7) and a Trehalose 6-Phosphate Phosphatase 10 (OsTPP10), respectively, known to be involved in anoxia and salt stress response. Members of the OsTPPs family playing a role in the Trehalose-6P (T6P)/SnRK1 system that regulates the C-metabolism under stress conditions. Indeed, high levels of T6P inhibit the cell energetic metabolism that is, on the contrary, stimulated when T6P is dephosphorylated by TPPs activities. The possible involvement of OsTPP7/10 in this regulative network and in the different salt sensitiveness of the different rice accessions has been investigated in two japonica accessions Olcenengo and SR113, salt tolerant and sensitive, respectively. The results obtained indicate that under salt condition (NaCl 150 mM) the coleoptile growth rate is less affected in Olcenengo where the T6P levels are quite lower than in SR113. The rate of growth in the controls appeared after 24 h from sowing and it was greater in Olcenengo than in SR113. Salt stress reduces coleoptile growth rate in each genotype but in SR113 the effect was more marked. In salt condition, Olcenengo showed a higher and earlier OsTPP10 expression than SR113; this could trigger SnRK1 activity and thus the mobilization of starch (α-amylase activity), supporting the energy needed for seed germination and coleoptile elongation under salt stress. The data confirmed that in the general framework of stress tolerance, members of the OsTPPs family play a key role to overcome saline stress during the first stages of germination. Moreover, on the basis of GWAS carried out on SES parameter, a QTL including a few genes that in the indica rice genome are localized within the major salinity tolerance-related QTL ‘SalTol’ have been identified. Among them, the gene encoding the vacuolar H+-pyrophosphatase 6 (OsOVP6) is present. Since OsOVP6 activity is suggested to be important in the network of the transport activities concerning Na+ and K+ transmembrane movement related to plant salt stress susceptibility/tolerance, an in depth physiological approach evaluating this possibility has been conducted. Two japonica salt-tolerant varieties (Galileo and Virgo), one japonica rice variety (PL12) known to be salt-susceptible, and the salt-tolerant indica genotype FL478 (containing the ‘SalTol’ QTL) have been considered. The Na+/K+ ratio, Na+ influx and K+ efflux, H+ extrusion, cytosolic and vacuolar pH by in vivo 31P-NMR techniques were evaluated in roots. The results obtained, together with the electrophysiological evaluation of the whole root Na+ conductance, allow to define a picture that may explain the different salt tolerance observed among the genotypes analyzed. In this picture, OsOVP6 plays a central role. In general, good variability within the population was observed for all the analyzed parameters. Salt stress tolerance is a multi-genic complex trait, and GWAS has proven to be a powerful tool for detecting natural variation underlying complex traits in crops. Through association analysis, several MTAs have been highlighted, with the identification of interesting loci, involved in the salt stress response. In particular, the idea of the function of two genes belonging to the OsTPPs family has been reinforced, confirming that in the general framework of salt tolerance, OsTPPs play a key role to overcome the stress during the germination stages. In this context, two varieties with contrasting behavior have been identified within the collection, Olcenengo and SR113, very tolerant and susceptible, respectively. Moreover, the idea of the vacuolar H+-pyrophosphatase function has been reinforced, too, since OsOVP6 activity is suggested to be important in the network of the transport activities concerning Na+ and K+ transmembrane movement related to plant salt stress tolerance. Finally, by evaluating the overall performance of each genotype of the population in relation to each parameter measured along the two-years experiment, a "core collection" of the 5 most tolerant and the 5 most susceptible varieties has been identified.