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 M₂ plants 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.

Keywords: AHAS; EMS Mutagenesis; Herbicide tolerance; Imazethapyr; Rice.

Fig. 1

Overall view of imazethapyr treated field and identification of resistant plant (inset) in M₂ generation

Fig. 2

Mutant progenies 15 days after imazethapyr treatment in M₄ generation. a: HTM-N22 (25th Day after sowing and at the time of treatment); b: HTM-N22 emerging from the weeds 15 days after treatment; c: Efficacy of the weed control (20th day after treatment); d: Nagina 22 killed along with the weeds

Fig. 3

F₂ population of HTM-N22-/Pusa 1656 tested by BSA with polymorphic SSR marker, RM 6844 in the vicinity of candidate gene, AHAS on Chromosome 2

Fig. 4

A representative gel picture showing the segregation pattern of RM 6844 in the F₂ mapping population from the cross, Pusa 1656/HTM-N22. M -50 bp Ladder, SP – Herbicide susceptible parent, Pusa 1656; TP- Herbicide tolerant mutant, HTM-N22; 1– 46 progenies in F₂ population

Fig. 5

Amino acid sequence alignment of AcetoHydroxy Acid Synthase (LOC_Os02g30630) between wild type Nagina 22 and herbicide tolerant mutant along with Nipponbare

Fig. 6

Line ribbon representation of the homology based protein model of WT (a) and HTM-N22 (b)