Application of agarose tilling to screen for mutations in stress-related genes in an IR64 mutant population

• Published in: Philippine Journal of Crop Science (Philippines) Vol. 32; no. 1
• Main Authors: Subramaninam, G., International Rice Research Inst., Los Banos, Laguna (Philippines). Plant Breeding, Genetics and Biotechnology Div, Wang Chuang, Zhejiang Univ., Huangzhou (China), Tharreau, D., UMR BgPL AGRO M-CIRAD-INRA, Montpellier (France), Albar, L., UMR 5096, IRD/CNRS/Universite de Perpignan, BP 64501, 34394 Montpellier CEDEX 5 (France), Shou Huixia, Zhejiang Univ., Huangzhou (China), Leung, H., International Rice Research Inst., Los Banos, Laguna (Philippines). Plant Breeding, Genetics and Biotechnology Div
• Format: Journal Article
• Language: English
• Published: 01.06.2007
• Subjects: GENE; GENES; GENETIC POLYMORPHISM; http://www.fao.org/aos/agrovoc#c_24031; http://www.fao.org/aos/agrovoc#c_3214; http://www.fao.org/aos/agrovoc#c_5013; http://www.fao.org/aos/agrovoc#c_5014; http://www.fao.org/aos/agrovoc#c_5259; http://www.fao.org/aos/agrovoc#c_5438; http://www.fao.org/aos/agrovoc#c_8157; MUTACION; MUTANT; MUTANTES; MUTANTS; MUTATION; NUCLEOTIDE; NUCLEOTIDES; NUCLEOTIDOS; ORYZA SATIVA; POLIMORFISMO GENETICO; POLYMORPHISME GENETIQUE; VARIEDADES; VARIETE; VARIETIES
• ISSN: 0115-463X

In this study, the author's aim was to identify single nucleotide polymorphism (SNPs) in the stress related candidate genes using Targeted Induced Local Lesions In Genome (TILLING) in the EMS-induced IR64 mutant population in rice. A set of 19 primer pairs specific for genes associated with yellow mottle virus, resistance, blast resistance, heat shock factors and kinases was chosen for the analysis.About 2000 M2 plants were raised and equal quantity of leaf samples from each eight plants were harvested together to form a pool. Heteroduplex was detected by the simplified method as described by Raghavan et al. 2007 Mol Breeding 19:87-101. From screening approximately 2000 M2 plants, two SNPs were detected in the yellow mottle virus resistance (RYMV1) gene, one of them changing the amino acid leucine to phenylalanine, while the other caused a silent mutation with no change in amino acid. Out of the two SNPs detected for the Pi 33 gene associated with blast resistance, one resulted in mis-sense mutation changing asparagine to aspartic acid, while the other was a silent mutation. A total of 10 SNPs were detected in genes associated with heat shock factors, transcription factors and kinases. Out of these, three SNPs caused mis-sense mutations changing the amino acids from aspartic acid to asparagine, glycine to glutamic acid and glycine to serine, respectively. All these SNPs were G/C to A/T transitions consistent with that expected from EMS mutagenesis. However, the estimated mutation density for this population was low (about one per MB). The above five mutants causing mis-sense mutation will be analyzed for phenotypic changes associated with the respective traits. Results suggests that the simple agarose TILLING is effective but a population with higher mutation density is needed to increase the efficiency of mutation detection.