MSc

The study entitled “Identification of Simple Sequence Repeat (SSR) markers linked to high
temperature tolerance in rice (Oryza sativa L.) by bulked segregant analysis” was conducted at
the Department of Molecular Biology and Biotechnology and the Department of Plant
Physiology, College of Agriculture, Vellayani, Thiruvananthapuram during 2022-23. The
objective of the study was to evaluate the F3 population of Uma X NERICA-L-44 for identifying
SSR markers linked to high temperature tolerance in rice by bulked segregant analysis and to
establish the role of the genes associated with them in heat tolerance.
Seeds collected from 46 F2 segregants (23 tolerant and 23 susceptible) of Uma X NL-44 along
with their parents were raised and transplanted to pots after 18 days of sowing in
the Kharif season of 2022. These 48 lines were kept under normal conditions up to the maximum
tillering stage and then transfered to polyhouse conditions where it was subjected to a high
temperature of 38-40 ⁰C. Phenotypic evaluation was done for plant height, tiller number,
productive tiller number, days to flowering, time of anthesis, Pollen viability, Spikelet fertility,
and 100 seed weight. Based on spikelet fertility percentage, 10 extremely tolerant and 10
extremely susceptible lines were selected.
DNA was extracted from the selected 10 heat tolerant and 10 susceptible lines along with the
parents by the modified Cetyltrimethylammoniumbromide (CTAB) method of DNA extraction.
The quality and quantity of extracted DNA were checked by using agarose gel electrophoresis
and spectrophotometric analysis. The DNA samples were screened by using 55 SSR primers
distributed across the rice genome. Out of 55 SSR primers, 18 of them showed polymorphism
between the parents. Then equal quantity of DNA was pooled to make heat tolerant and
susceptible bulks. The bulked DNA samples were screened using the 18 SSR primers that have
shown polymorphism between parents. The polymorphic markers between the tolerant and
susceptible bulks were used to study the segregation of the alleles in the individual lines
constituting the tolerant and susceptible bulks. Through bulked segregant analysis (BSA), 10
SSR markers were found polymorphic between tolerant and susceptible bulks and their
individual lines. It revealed the possible presence of genetic loci for heat tolerance in those
locations. Out of the 10 SSR markers identified in the BSA (RM337, RM10793, RM242,
RM5749, RM6100, RM490, RM3475, RM470, RM473, and RM556), nine of the markers have
been previously reported for heat tolerance traits. RM337 is newly identified in the present study.
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The genes in the 200 kb vicinity of the RM markers were retrieved from the Rice Annotation
Project database. To get a deeper insight into how these genes participate in heat tolerance, gene
annotation, gene ontology (GO) enrichment analysis, and trait ontology (TO) were performed for
all the significant markers. Upon screening of the loci in the proposed region, genes LOP1
(LOC_Os08g01330) and LOP2 (LOC_Os08g0112) were found to be associated with RM337.
LOP1 is a NAC transcription factor that is reported to be involved in the regulation of cellulose
synthesis, secondary wall biosynthesis (Os08t0103900-01), and inflorescence development.
LOP2 is also known as OsMOT1, which is a molybdate transporter involved in the uptake and
translocation of molybdate (Os08t0101500-01). Hence their expression was analyzed in the two
rice varieties under both control and high temperature conditions. LOP1 was found to be
significantly upregulated in the NL-44 variety under high temperature condition compared to the
normal temperature conditions and susceptible variety, Uma. On the other hand, the gene LOP2
was found to be upregulated in both varieties under the higher temperature condition compared
to their respective controls. However, the relative expression in Uma was higher than in NL-44.
In the present study, the phenotypic evaluation and bulked segregant analysis using 55 SSR
primers in F3 generation of Uma X NL-44 revealed that 10 SSR markers (RM222, RM242,
RM337, RM470, RM473, RM490, RM556, RM5749, RM6100, RM10793) are linked to high
temperature tolerance in rice. The newly identified SSR marker RM337 and associated gene
LOP1 is also linked to high temperature tolerance in rice. The results demonstrate that BSA
using SSR markers and gene annotation and enrichment analysis is useful in identifying genomic
regions and genes that contribute to thermotolerance respectively. Also, these F3 lines can be
used for the development of high temperature tolerant rice varieties and these markers can be
used for marker assisted selection (MAS) in rice

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