Whole transcriptome assembly and annotation of wild wheat Aegilops speltoides for identification of novel heat stress tolerance genes
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Date
2021
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Punjab Agricultural University, Ludhiana
Abstract
Wheat, a major staple food crop of the world, is relatively sensitive to the changing climatic
conditions, especially the high temperature. The comprehensive mechanism of heat stress
response at the molecular level and exploitation of candidate heat tolerant genes in the wild
species of wheat Aegilops speltoides have not been exclusively studied. Using transcriptome
data, we made comparative RNA-Seq analysis for the gene expression profiles of heat stress
tolerant genes in diploid wheat Aegilops speltoides acc. pau3809, tetraploid wheat Triticum
durum cv. PDW274 and hexaploid wheat Triticum aestivum cv. PBW725. cDNA libraries
generated from flag leaf tissues of AS3809, PDW274 and PBW725 were sequenced using
Illumina technology and approx. 22M, 10M and 14M high-quality clean reads were obtained
respectively. De novo assembly was generated for all three genotypes to avoid any bias and
we obtained 94,946; 68,873 and 76,404 genes and N50 value of 1409, 904 and 998 bp for
AS3809, PDW274 and PBW725 transcriptome respectively. Annotations were performed
using BLAST2GO, PfamScan and MapMan and the identified genes in the three wheat
transcriptomes were divided into 53 GO functional categories under the biological process,
molecular function and cellular component. OrthoMCL was used to find ortholog transcripts
across the three wheat genotypes. In this investigation, we analyzed the expression abundance
of heat shock transcription factors (HSFs) which are known for their roles in plant tolerance
to abiotic stresses. 12 HSF orthologs classified to class A, B and C were studied for
comparative expression abundance in the three wheat transcriptomes. The expression of all
the HSFs under study was observed to be highest in AS3809, followed by PDW274 and least
expression was observed in PBW725. We validated 8 HSFs under control and heat stressed
condition in the three wheat genotypes using qRT-PCR. Six out of eight HSF genes showed
higher expression values in AS3809 sample (both in control and HS) as compared to the other
two genotypes which corroborates with the expression values obtained from the transcriptome
data. Our results demonstrate that the HSFs have higher gene expression in the wild wheat
Aegilops speltoides as compared the cultivated wheat and they play positive roles in plant
tolerance to heat stress. The HSFs from Aegilops speltoides may be used as a candidate gene
for genetic modification of cool-season plant species for improving heat tolerance.
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Citation
Seni, Sushmita (2021). Whole transcriptome assembly and annotation of wild wheat Aegilops speltoides for identification of novel heat stress tolerance genes (Unpublished M.Sc. thesis). Punjab Agricultural University, Ludhiana, Punjab, India.