PhD

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
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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
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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.