Development and Genetic Characterization of A Novel Herbicide (Imazethapyr) Tolerant Mutant in Rice (Oryza sativa L.)

Abstract

BackgroundIncreased 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.ResultsWe 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.ConclusionsA 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12284-017-0151-8) contains supplementary material, which is available to authorized users.

Inheritance and Molecular Characterization of a Novel Mutated AHAS Gene Responsible for the Resistance of AHAS-Inhibiting Herbicides in Rapeseed (Brassica napus L.)

“…Finding new nontransgenic herbicide-resistant rapeseed materials is necessary due to the limitation of herbicide-resistant cultivars with independent intellectual property rights and the prohibition of commercial production of transgenic rapeseed in China. At present, many other crops are found harboring the AHAS-inhibiting herbicide resistance by nontransgenic ways due to the single-point mutation in AHAS genes [25,30,31]. In addition, several rapeseed mutants conferring the AHAS-inhibiting herbicide resistance have been reported and proven due to mutations in BnAHASs, such as BnAHAS1-S653A [6,24], BnAHAS3-S197L [21] and BnAHAS3-T574L [24,32].…”

Exploring natural allelic variations of the β-triketone herbicide resistance gene HIS1 for application in indica rice and particularly in two-line hybrid rice

“…Although 2-line hybrid rice has the advantages of simpler breeding procedures and more freedom in combination, the P/TGMS sterile lines are negatively affected by light and temperature during seed production, resulting in self-crossing, which seriously affects the quality of hybrid seeds. Previous studies have attempted to create herbicide-sensitive sterile lines or herbicide-resistant restorer lines through genetic modi cation and physical/chemical mutagenesis (Shoba et al, 2017;Tang et al, 2020;Xia et al, 2019;Zhang et al, 2002;Zhang et al, 2014), but until now, they have not solved the seed purity problem of 2-line hybrid rice at a large scale except for the use of Imidazolinone-resistant restorers in the United States (Sudianto et al, 2013). In this study, we identi ed eight 2-line sterile lines naturally sensitive to bTHs, whereas many of the corresponding restorer lines were resistant.…”

Push It to the Limit: Identification of Novel Amino Acid Changes on the Acetolactate Synthase Enzyme of Rice That Putatively Confer High Level of Tolerance to Different Imidazolinones

“…double and triple mutations for Imazapyr were Arg351/Ser627 and Phe180/Arg351/Ser627, respectively, while the quadruple mutation Val170/Phe180/Arg351/Ser627 decreased 44% the estimated free energy of binding when compared to the wild type enzyme ( Table 2). Most of the Imidazolinone-tolerant rice cultivars are currently developed from either one or a combination of Ala179, Trp548, and Ser627 mutations (Tan et al, 2005;Shoba et al, 2017;Piao et al, 2018;Li et al, 2019), with the combination Trp548 and Ser627 being the most recurrent. It is interesting to note that the most effective double and triple mutations detected in our analysis to the four Imidazolinones encompass only one of the classical mutations (Trp548 for Imazapic, Imazaquin, and Imazethapyr; Ser627 for Imazapyr), while the other putative mutations are novel and could be even more effective (Lys230 for Imazapic, Imazaquin, and Imazethapyr; Arg351 for Imazapyr).…”

“…The mechanism of action of herbicides are associated to the modulation of enzymes that are essential for a plant's growth and survival. In addition, among a vast number of herbicides, the class of Sulfonylureas and Imidazolinones are the most used to develop new tolerance crops, since it is already know how these molecules act in plants (Endo and Toki, 2013;Shoba et al, 2017). The development of herbicide tolerant plants have been achieved by introducing specific mutations (non-GM approach) in the target site of herbicide action (Green and Owen, 2011), since it has the advantage of easier registration/release for commercial cultivation as well as wider public acceptance (Fartyal et al, 2018).…”

“…The development of herbicide tolerant plants have been achieved by introducing specific mutations (non-GM approach) in the target site of herbicide action (Green and Owen, 2011), since it has the advantage of easier registration/release for commercial cultivation as well as wider public acceptance (Fartyal et al, 2018). Among different kind of herbicides, Imidazolinones are probably the most widely targeted ones for developing herbicide tolerant crops through non-GM approach (Shoba et al, 2017).…”