Browsing by Author "SENTHILKUMAR, K. M."

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    Gene discovery and allele mining for thermotolerance in wheat using transcriptome analysis
    (National Research Centre on Plant Biotechnology Indian Agricultural Research Institute New Delhi –, 2017) SENTHILKUMAR, K. M.; K. C. Bansal
    Wheat is an important staple food and its cultivation is important for global food security. Due to global climate change, high temperature is emerging as a major threat to wheat productivity in India. It is important to identify novel genes and promoters for high temperature stress (HS) tolerance to breed climate resilient wheat genotypes. Many genes have been functionally validated for HS tolerance in crop plants. Fewer efforts have been made to study their orthologs in wheat. It is a polyploid with homeologous chromosomes, which still remains a challenge for genomic studies, and isolation and characterization of agronomically important genes. Recent advances in Next Generation Sequencing (NGS) technologies and high-throughput bioinformatics analysis overcame these barriers and made it possible to generate various genomic resources for wheat. In this study, we utilized these advance genomics tools to identify and mine the alleles of HS responsive genes in wheat genotypes with contrasting thermotolerance. Our analysis lead to the identification of ~250 HS responsive genes in wheat including 9 members of Caseinolytic protease (Clp) family, 163 members of Small Heat Shock Protein (sHSP)/TaHSP20 family and 37 HS responsive genes (excluding HSPs and HSF). Phylogenetic analysis of Clp and TaHSP20 family proteins with sequenced grass genomes (Oryza sativa, Sorghum bicolor, Zea mays, Brachypodium distachyon and Setaria italica) showed that these HS responsive genes were highly conserved across grass genomes. Spatio-temporal and abiotic stress specific expression pattern in normalized wheat array datasets revealed constitutive as well as inductive response of TaClps and TaHSPs in different tissues and developmental stages. Expression analysis revealed that TaClpB2 and TaClpB3 genes were upregulated under heat, salt and oxidative stress but were downregulated by cold stress in most genotypes. TaClpC1, TaClpC2 and TaClpD1 genes were significantly upregulated by cold stress in some of the wheat genotypes. TaClpD2 gene was upregulated only in DBW16 under salt stress. Small HSPs (sHSPs) play crucial role in protecting cellular macromolecules and function under heat stress, and thus contribute to thermotolerance. Hence, expression pattern of 13 TaHSP20 genes were analyzed in heat and drought tolerant wheat genotype C306. Among these genes, TaHSP16.8 exhibited ii constitutively, while TaHSP17.4, TaHSP17.6, TaHSP19.1, TaHSP19.9, TaHSP20.6, TaHSP23.6 were significantly induced by heat stress. TaHSP16.8, TaHSP17.4, TaHSP17.6, TaHSP19.1, TaHSP19.9 and TaHSP20.6 were also induced under salt stress. Except TaHSP16.8, none of the sHSPs showed expression under cold stress. The results suggests that TaHSP17.4, TaHSP17.6, TaHSP19.1, TaHSP19.9, TaHSP20.6 may provide tolerance to both heat and salt stresses. TaHSP16.8 appears to be functioning specifically under temperature extremes, while TaHSP23.6 may play specific protective role under salt stress. In addition to the HSP genes analyzed, 37 heat stress induced genes (excluding HSPs and HSFs) were also identified from wheat and their expression pattern under different abiotic stresses were characterized. TaFES1A, TaFAD8 and TaCPR5 expression were increased under heat, cold and salt stress conditions. TaAPX2, TaDREB2a and TaHSA32 expression were increased under heat and salt stress. TaCBK3 expression was increased under cold and salt stress. TaMBF1c expression was increased under heat stress, whereas TaHIT1 expression was increased under cold stress. Five HS responsive genes namely TaHSA32, TaFAD8, TaCPR5, TaBI and TaDREB2a having diverse cellular functions were mined in a set of 64 wheat genotypes having contrasting thermotolerance. Seven genotypes namely Stiletto, Warigal, WL 711, Kalgarin, Arena, Kharchiya local and Luan displayed one SNP at 379th position, a A to C transversion and one dinucleotide insertion CA at 411th position in TaHSA32 gene. The genotype Wilgoyne has one SNP at 660th position, a T to C transversion in TaDREB2A gene. However, this mutation doesn’t changed the amino acid sequences of TaHSA32 and TaDREB2A. TaDREB2a and TaHSA32 are highly conserved among wheat genotypes, whereas TaFAD8 and TaBI-1 displayed higher sequence difference. Out of 41 candidate gene based SSRs (cg-SSRs), one SSR from TaCHIP-1 gene displayed polymorphism among the seven genotypes differing in thermotolerance. Our results showed that the identified high temperature responsive genes play important role in imparting HS tolerance and also plays distinct roles in response to different abiotic stress conditions. The data obtained from this study contribute to a better understanding of the complexity of the high temperature responsive genes in wheat, and provide the basis for further studies to dissect the function of these genes during plant growth and development as well as in response to environmental stimuli.