High temperature medicated changes in sugar signaling pathway and identification of associated microsatellite markers in rice (Oryza sativa L.)

Abstract

The research work entitled ‘High temperature mediated changes in sugar signaling pathway and identification of associated microsatellite markers in rice (Oryza sativa L.)’ was undertaken at the College of Agriculture, Vellayani during 2018-2022. The aim of the study was to understand the effect of high temperature stress on the changes in the sugar signaling pathway and to identify the molecular markers associated with heat tolerance in rice. The investigation was carried out as four experiments, the first being the study of heat stress effects on the sugar signaling pathway, and the remaining three experiments being interconnected. The second experiment was the laying of crossing blocks to produce F1 and F2 generation seeds, after which the third experiment was conducted to phenotypically evaluate the F2 population derived from the second experiment. The fourth experiment was conducted to identify polymorphic micro-satellite markers that were associated with heat tolerance in the F2 population evaluated in the previous experiment. In the first experiment, the genotypes NERICA L-44 (NL-44) and Vandana were evaluated under two different temperature conditions viz. high temperature stress (38-42 ˚C) and ambient (26-34 ˚C) which was taken as control. The expression of four genes viz. OsHXK2, OsSnRK1, OsTOR and OsTPS1 was studied using quantitative real-time polymerase chain reaction (qRT-PCR) at the vegetative phase just before panicle initiation and at the grain filling stage. The results of the experiment showed that under high temperature stress conditions, the performance of the variety NL-44 was superior to the variety Vandana, as measured by parameters such as cell membrane stability index (+10%), pollen viability (+19%), panicle length (+4.8 cm), photosynthetic rate (+4.75 µmol cm-2 s - 1 ), stomatal conductance (+0.027 mol m-2 s -1 ), spikelet fertility (+19.1%), 1000 grain weight (+5.5 g) as well as greater photochemical efficiency (Fv/Fm ratio), maximal quantum yield (ΦPSII), electron transport rate and higher water-use efficiency. The results of the experiment clearly reinforce the tolerance characteristics of NL-44 and establish the susceptibility of Vandana to high temperature stress. The expression of each gene was correlated with multiple traits that explained the tolerance or susceptibility of the genotypes 204 under heat stress. Based on such correlations, the proposed pathway of sugar signaling in tolerant rice genotypes should necessarily cause upregulation of OsHXK2, as it indicates high glucose content; down-regulation of OsSnRK1, as it would prevent the induction of catabolic processes; up-regulation of OsTOR, as it indicates higher nutrient status as well as being involved in inducing stress responses; and down-regulation of OsTPS1 as the low T6P content would signal remobilization of nutrients towards stress response. In the second experiment, the two varieties NL-44 (heat-tolerant) and the high yielding Uma (heat-susceptible) were crossed to produce F1 generation hybrids. The F1 seeds were then selfed to produce F2 generation seeds. The F2 seeds produced had morphological characters that were intermediate to the parents with medium length and medium boldness unlike NL-44 which was long and slender grain whereas Uma has characteristic short and bold grain characteristics. 144 F2 plants along with the parents, NL-44 and Uma were phenotypically evaluated under high temperature stress (36-40 ˚C) in the third experiment. . The mean of the population was closer to NL-44 with regards to the traits of plant height, membrane stability index, photosynthetic rate, stomatal conductance, transpiration rate, pollen viability, spikelet fertility and 1000 grain weight. However, for the traits such as tiller number, days to flowering, time of anthesis, leaf temperature and panicle length, the mean of the population was influenced by the susceptible parent, Uma. Based upon the spikelet fertility percentage, the ten most tolerant and ten most susceptible plants were selected from the F2 plants and Bulked Segregant Analysis (BSA) technique was used to identify polymorphic SSR markers that are linked to high temperature tolerance. The results of the study have identified 18 SSR markers that exhibited polymorphism between the parents out of the 100 SSR markers used. The identified polymorphic markers could distinguish between the tolerant and susceptible bulks as seen in their differential banding pattern. The identified polymorphic markers were also found to segregate between the individual lines and characterize them into tolerant or 205 susceptible lines based on their similarity to the banding pattern of the tolerant parent, NL44 or the susceptible parent, Uma. In the present study, the expression levels of the sugar signaling genes and their association with phenotypic characters has elucidated their role in imparting heat tolerance in rice. The phenotyping of the F2 generation indicated that the tolerance traits in the population were majorly contributed by the tolerant parent i.e. NL-44. The identified polymorphic markers were able to segregate the individual lines of F2 population into tolerant and susceptible genotypes. The elucidation of the sugar signaling mechanism in tolerant genotypes of rice and the association of molecular markers linked to the heat tolerance trait in the segregating second generation filial populations is validated to be beneficial in undertaking crop improvement studies for enhanced sugar metabolism as well as to introgress the tolerance traits into high-yielding regional varieties.