title: Development and Genetic Characterization of A Novel Herbicide (Imazethapyr) Tolerant Mutant in Rice (Oryza sativa L.).
abstract: BACKGROUND
  Increased water and labour scarcity in major rice growing areas warrants a shift towards direct seeded rice cultivation under which management of weeds is a major issue. Use of broad spectrum non-selective herbicides is an efficient means to manage weeds. Availability of rice genotypes with complete tolerance against broad-spectrum non-selective herbicides is a pre-requisite for advocating use of such herbicides. In the present study, we developed an EMS induced rice mutant, 'HTM-N22', exhibiting tolerance to a broad spectrum herbicide, 'Imazethapyr', and identified the mutations imparting tolerance to the herbicide.
  
  
  RESULTS
  We identified a stable and true breeding rice mutant, HTM-N22 (HTM), tolerant to herbicide, Imazethapyr, from an EMS-mutagenized population of approximately 100,000 M2plants of an upland rice variety, Nagina 22 (N22). Analysis of inheritance of herbicide tolerance in a cross between Pusa 1656-10-61/HTM showed that this trait is governed by a single dominant gene. To identify the causal gene for Imazethapyr tolerance, bulked segregant analysis (BSA) was followed using microsatellite markers flanking the three putative candidate genes viz., an Acetolactate Synthase (ALS) on chromosome 6 and two Acetohydroxy Acid Synthase (AHAS) genes, one on chromosomes 2 and another on chromosome 4. RM 6844 on chromosome 2 located 0.16 Mbp upstream of AHAS (LOC_Os02g30630) was found to co-segregate with herbicide tolerance. Cloning and sequencing of AHAS (LOC_Os02g30630) from the wild type, N22 and the mutant HTM and their comparison with reference Nipponbare sequence revealed several Single Nucleotide Polymorphisms (SNPs) in the mutant, of which eight resulted in non-synonymous mutations. Three of the eight amino acid substitutions were identical to Nipponbare and hence were not considered as causal changes. Of the five putative candidate SNPs, four were novel (at positions 30, 50, 81 and 152) while the remaining one, S627D was a previously reported mutant, known to result in Imidazolinone tolerance in rice. Of the novel ones, G152E was found to alter the hydrophobicty and abolish an N myristoylation site in the HTM compared to the WT, from reference based modeling and motif prediction studies.
  
  
  CONCLUSIONS
  A novel mutant tolerant to the herbicide "Imazethapyr" was developed and characterized for genetic, sequence and protein level variations. This is a HTM in rice without any IPR (Intellectual Property Rights) infringements and hence can be used in rice breeding as a novel genetic stock by the public funded organizations in the country and elsewhere.
authors.affiliation: Tamil Nadu Agricultural University, Coimbatore, 641003, India.
authors.forename: D
authors.initials: D
authors.lastname: Shoba
authors.affiliation: Tamil Nadu Agricultural University, Coimbatore, 641003, India.
authors.forename: M
authors.initials: M
authors.lastname: Raveendran
authors.affiliation: Tamil Nadu Agricultural University, Coimbatore, 641003, India.
authors.forename: S
authors.initials: S
authors.lastname: Manonmani
authors.affiliation: Tamil Nadu Agricultural University, Coimbatore, 641003, India.
authors.forename: S
authors.initials: S
authors.lastname: Utharasu
authors.affiliation: Tamil Nadu Agricultural University, Coimbatore, 641003, India.
authors.forename: D
authors.initials: D
authors.lastname: Dhivyapriya
authors.affiliation: Tamil Nadu Agricultural University, Coimbatore, 641003, India.
authors.forename: G
authors.initials: G
authors.lastname: Subhasini
authors.affiliation: Tamil Nadu Agricultural University, Coimbatore, 641003, India.
authors.forename: S
authors.initials: S
authors.lastname: Ramchandar
authors.affiliation: Tamil Nadu Agricultural University, Coimbatore, 641003, India.
authors.forename: R
authors.initials: R
authors.lastname: Valarmathi
authors.affiliation: Division of Genetics, ICAR-Indian Agricultural Research Institute, New Delhi, 110012, India.
authors.forename: Nitasha
authors.initials: N
authors.lastname: Grover
authors.affiliation: Division of Genetics, ICAR-Indian Agricultural Research Institute, New Delhi, 110012, India.
authors.forename: S Gopala
authors.initials: SG
authors.lastname: Krishnan
authors.affiliation: Division of Genetics, ICAR-Indian Agricultural Research Institute, New Delhi, 110012, India.
authors.forename: A K
authors.initials: AK
authors.lastname: Singh
authors.affiliation: ICAR-National Research Centre on Plant Biotechnology, Pusa, New Delhi, 110012, India.
authors.forename: Pawan
authors.initials: P
authors.lastname: Jayaswal
authors.affiliation: ICAR-National Research Centre on Plant Biotechnology, Pusa, New Delhi, 110012, India.
authors.forename: Prashant
authors.initials: P
authors.lastname: Kale
authors.affiliation: ICAR-National Research Centre on Plant Biotechnology, Pusa, New Delhi, 110012, India.
authors.forename: M K
authors.initials: MK
authors.lastname: Ramkumar
authors.affiliation: ICAR-National Research Centre on Plant Biotechnology, Pusa, New Delhi, 110012, India.
authors.forename: S V Amitha
authors.initials: SVA
authors.lastname: Mithra
authors.affiliation: Indian Council of Agriculture Research, New Delhi, 110 001, India.
authors.forename: T
authors.initials: T
authors.lastname: Mohapatra
authors.affiliation: Punjab Agricultural University, Ludhiana, 141004, India.|Present address: ICAR-National Bureau of Plant Genetic Resources, Pusa, New Delhi, 110012, India.
authors.forename: Kuldeep
authors.initials: K
authors.lastname: Singh
authors.affiliation: ICAR-National Research Centre on Plant Biotechnology, Pusa, New Delhi, 110012, India.
authors.forename: N K
authors.initials: NK
authors.lastname: Singh
authors.affiliation: Indian Institute of Rice Research, Rajendranagar, Hyderabad, 500030, India.
authors.forename: N
authors.initials: N
authors.lastname: Sarla
authors.affiliation: University of Agricultural Sciences, Bengaluru, 560065, India.
authors.forename: M S
authors.initials: MS
authors.lastname: Sheshshayee
authors.affiliation: National Rice Research Institute, Cuttack, Odisha, 753006, India.
authors.forename: M K
authors.initials: MK
authors.lastname: Kar
authors.affiliation: Tamil Nadu Agricultural University, Coimbatore, 641003, India. robin.tnau@gmail.com.
authors.forename: S
authors.identifier: http://orcid.org/0000-0002-1830-1843
authors.initials: S
authors.lastname: Robin
authors.affiliation: INSA Honorary Scientist, NRCPB, IARI, Pusa, New Delhi, 110012, India.
authors.forename: R P
authors.initials: RP
authors.lastname: Sharma
keywords: AHAS; EMS Mutagenesis; Herbicide tolerance; Imazethapyr; Rice