Mariano Bulos

Molecular and biochemical characterization of an induced mutation conferring imidazolinone resistance in sunflower.

A partially dominant nuclear gene conferring resistance to the imidazolinone herbicides was previously identified in the cultivated sunflower (Helianthus annuus L.) line CLHA-Plus developed by seed mutagenesis. The objective of this study was to characterize this resistant gene at the phenotypic, biochemical and molecular levels. CLHA-Plus showed a complete susceptibility to sulfonylureas (metsulfuron, tribenuron and chlorsulfuron) but, on the other hand, it showed a complete resistance to imidazolinones (imazamox, imazapyr and imazapic) at two rates of herbicide application. This pattern was in close association with the AHAS-inhibition kinetics of protein extracts of CLHA-Plus challenged with different doses of imazamox and chlorsulfuron. Nucleotide and deduced amino acid sequence comparisons between resistant and susceptible lines indicated that the imidazolinone-resistant AHAS of CLHA-Plus has a threonine codon (ACG) at position 122 (relative to the Arabidopsis thalianaAHAS sequence), whereas the herbicide-susceptible enzyme from BTK47 has an alanine residue (GCG) at this position. Since the resistance genes to AHAS-inhibiting herbicides so far characterized in sunflower code for the catalytic (large) subunit of AHAS, we propose to redesignate the wild type allele as ahasl1 and the incomplete dominant resistant alleles as Ahasl1-1 (previously Imr1 or Arpur), Ahasl1-2 (previously Arkan) and Ahasl1-3 (for the allele present in CLHA-Plus). The higher tolerance level to imidazolinones and the lack of cross-resistance to other AHAS-inhibiting herbicides of Ahasl1-3 indicate that this induced mutation can be used to develop commercial hybrids with superior levels of tolerance and, at the same time, to assist weed management where control of weedy common sunflower is necessary.

Inheritance and molecular characterization of broad range tolerance to herbicides targeting acetohydroxyacid synthase in sunflower

Ahasl1 is a multilallelic locus where all the induced and natural mutations for herbicide tolerance were described thus far in sunflower (Helianthus annuus L.). The allele Ahasl1-1 confers moderate tolerance to imidazolinone (IMI), Ahasl1-2, and Ahasl1-3 provides high levels of tolerance solely to sulfonylurea (SU) and IMI, respectively. An Argentinean wild sunflower population showing plants with high level of tolerance to either an IMI and a SU herbicide was discovered and used to develop an inbred line designated RW-B. The objectives of this work were to determine the relative level and pattern of cross-tolerance to different AHAS-inhibiting herbicides, the mode of inheritance, and the molecular basis of herbicide tolerance in this line. Slight or no symptoms observed after application of different herbicides indicated that RW-B possesses a completely new pattern of tolerance to AHAS-inhibiting herbicides in sunflower. Biomass response to increasing doses of metsulfuron or imazapyr demonstrated a higher level of tolerance in RW-B with respect to Ahasl1-1/Ahasl1-1 and Ahasl1-2/Ahasl1-2 lines. On the basis of genetic analyses and cosegregation test, it was concluded that tolerance to imazapyr in the original population is inherited as a single, partially dominant nuclear gene and that this gene is controlling the tolerance to four different AHAS-inhibiting herbicides. Pseudo-allelism test permitted us to conclude that the tolerant allele present in RW-B is an allelic variant of Ahasl1-1 and was designated as Ahasl1-4. Nucleotide and deduced amino acid sequence indicated that the Ahasl1-4 allele sequence of RW-B has a leucine codon (TTG) at position 574 (relative to the Arabidopsis thaliana AHAS sequence), whereas the enzyme from susceptible lines has a tryptophan residue (TGG) at this position. The utilization of this new allele in the framework of weed control and crop rotation is discussed.

Molecular characterization of Als1 , an acetohydroxyacid synthase mutation conferring resistance to sulfonylurea herbicides in soybean

Key messageThe AHAS gene family in soybean was characterized. The locusAls1for sulfonylurea resistance was mapped and the resistant allele was characterized at the molecular level.AbstractSulfonylurea (SU) resistance in soybean is controlled by Als1, a semi-dominant allele obtained by EMS mutagenesis over the cultivar Williams 82. The overall objective of this research was to map Als1 in the soybean genome and to determine the nucleotidic changes conferring resistance to SU. Four nucleotide sequences (GmAhas1–4) showing high homology with the Arabidopsis thaliana acetohydroxyacid synthase (AHAS, EC 4.1.3.18) gene sequence were identified by in silico analysis, PCR-amplified from the SU-resistant line BTK323STS and sequenced. Expression analysis showed that GmAhas1, located on chromosome 4 by in silico analysis, is the most expressed sequence in true leaves. F2:3 families derived from the cross between susceptible and resistant lines were evaluated for SU resistance. Mapping results indicate that the locus als1 is located on chromosome 4. Sequence comparison of GmAhas1 between BTK323STS and Williams 82 showed a single nucleotide change from cytosine to thymine at position 532. This transversion generates an amino acid change from proline to serine at position 197 (A. thaliana nomenclature) of the AHAS catalytic subunit. An allele-specific marker developed for the GmAhas1 mutant sequence cosegregated with SU resistance in the F2 population. Taking together, the mapping, expression and sequencing results indicate that the GmAhas1 sequence corresponds to the Als1 gene sequence controlling SU resistance in soybean. The molecular breeding tools described herein create the basis to speed up the identification of new mutations in soybean AHAS leading to enhanced levels of resistance to SU or to other families of AHAS inhibitor herbicides.

Phenotypic characterization, genetic mapping and candidate gene analysis of a source conferring reduced plant height in sunflower

Reduced height germplasm has the potential to increase stem strength, standability, and also yields potential of the sunflower crop (Helianthus annuus L. var. macrocarpus Ckll.). In this study, we report on the inheritance, mapping, phenotypic and molecular characterization of a reduced plant height trait in inbred lines derived from the source DDR. This trait is controlled by a semidominant allele, Rht1, which maps on linkage group 12 of the sunflower public consensus map. Phenotypic effects of this allele include shorter height and internode length, insensibility to exogenous gibberellin application, normal skotomorphogenetic response, and reduced seed set under self-pollination conditions. This later effect presumably is related to the reduced pollen viability observed in all DDR-derived lines studied. Rht1 completely cosegregated with a haplotype of the HaDella1 gene sequence. This haplotype consists of a point mutation converting a leucine residue in a proline within the conserved DELLA domain. Taken together, the phenotypic, genetic, and molecular results reported here indicate that Rht1 in sunflower likely encodes an altered DELLA protein. If the DELPA motif of the HaDELLA1 sequence in the Rht1-encoded protein determines by itself the observed reduction in height is a matter that remains to be investigated.

Molecular mapping of a sunflower rust resistance gene from HAR6

Sunflower rust, caused by Puccinia helianthi Schw., can result in significant yield losses in cultivated sunflower (Helianthus annuus L. var. macrocarpus Ckll.). HAR6 is a germplasm population resistant to most predominant rust races. The objectives of this study were to map the resistance factor present in HAR6 (RHAR6), and to provide and validate molecular tools for the identification of this gene for marker assisted selection purposes. Virulence reaction of seedlings for the F2 population and F2:3 families suggested that a single dominant gene confers rust resistance in HAR6-1, a selected rust resistance line from the original population. Genetic mapping with eight markers covered 97.4 cM of genetic distance on linkage group 13 of the sunflower consensus map. A co-dominant marker ZVG61 is the closest marker distal to RHAR6 at a genetic distance of 0.7 cM, while ORS581, a dominant marker linked in the coupling phase, is proximal to RHAR6 at a genetic distance of 1.5 cM. Validation of these markers was assessed by converting a susceptible line into a rust resistant isoline by means of marker assisted backcrossing. The application of these results to assist the breeding process and to design new strategies for rust control in sunflower is discussed.

Occurrence of the rust resistance gene Lr37 from Aegilops ventricosa in Argentine cultivars of wheat

Leaf rust of wheat ( Triticum aestivum L.) caused by the fungus Puccinia triticina (formerly P. recondita f. sp. tritici ), is one of the most important foliar diseases of this crop. Lr37 rust resistance gene, which confers resistance in wheat against leaf rust, was introgressed into cultivated wheat from Aegilops ventricosa Tausch. Rust races with virulence to Lr37 have been identified in different countries, but it still provides resistance to a wide range of races and is useful in combination with other resistance genes. There are no reports about the presence, frequency and origin of Lr37 in Argentinean wheat cultivars. In this work, we analyzed 88 registered Argentinean wheat cultivars developed by different breeding companies and institutions during the last 15 years by means of a molecular marker which is diagnostic of the 2NS-2AS translocation which carries Lr37 . Only 4 cultivars showed the amplification product associated with this chromosome fragment. These four cultivars which carry the translocated 2NS-2AS chromosome were registered by the same breeding company during the last seven years and all of them have European germplasm in their genealogy. To the best of our knowledge this is the first report of the presence of Lr37 in registered South American cultivars.

Genetic mapping, marker assisted selection and allelic relationships for the Pu6 gene conferring rust resistance in sunflower

Rust resistance in the sunflower line P386 is controlled by Pu6, a gene which was reported to segregate independently from other rust resistant genes, such as R4. The objectives of this work were to map Pu6, to provide and validate molecular tools for its identification, and to determine the linkage relationship of Pu6 and R4. Genetic mapping of Pu6 with six markers covered 24.8 cM of genetic distance on the lower end of linkage Group 13 of the sunflower consensus map. The marker most closely linked to Pu6 was ORS316 at 2.5 cM in the distal position. ORS316 presented five alleles when was assayed with a representative set of resistant and susceptible lines. Allelism test between Pu6 and R4 indicated that both genes are linked at a genetic distance of 6.25 cM. This is the first confirmation based on an allelism test that at least two members of the Radv/R4/R11/ R13a/R13b/Pu6 cluster of genes are at different loci. A fine elucidation of the architecture of this complex locus will allow designing and constructing completely new genomic regions combining genes from different resistant sources and the elimination of the linkage drag around each resistant gene.

Root biomass response to foliar application of imazapyr for two imidazolinone tolerant alleles of sunflower (Helianthus annuus L.).

Imisun and CLPlus are two imidazolinone tolerance traits in sunflower (Helianthus annuus L.) determined by the expression of two alleles at the locus Ahasl1. Both traits differed in their tolerance level to imazapyr —a type of imidazolinone herbicide— when aboveground biomass is considered, but the concomitant herbicide effect over the root system has not been reported. The objective of this work was to quantify the root biomass response to increased doses of imazapyr in susceptible (ahasl1/ahasl1), Imisun (Ahasl1-1/Ahasl1-1) and CLPlus (Ahasl1-3/Ahasl1-3) homozygous sunflower genotypes. These materials were sprayed at the V2–V4 stage with increased doses of imazapyr (from 0 to 480 g active ingredient ha−1) and 14 days after treatment root biomass of each plant was assessed. Genotype at the Ahasl1 locus, dose of imazapyr and their interaction significantly contributed (P < 0.001) to explain the reduction in root biomass accumulation after herbicide application. Estimated dose of imazapyr required to reduce root biomass accumulation by fifty percent (GR50) differed statistically for the three genotypes under study (P < 0.001). CLPlus genotypes showed the highest values of GR50, 300 times higher on average than the susceptible genotypes, and almost 8 times higher than Imisun materials, demonstrating that both alleles differ in their root biomass response to foliar application of increased doses of imazapyr.

Contribution of non-target-site resistance in imidazolinone-resistant Imisun sunflower

ABSTRACT: The first commercial herbicide-resistant trait in sunflower ( Helianthus annuus L.) is known as ‘Imisun’. Imidazolinone resistance in Imisun cultivars has been reported to be genetically controlled by a major gene (known as Imr1 or Ahasl1-1 ) and modifier genes. Imr1 is an allelic variant of the Ahasl1 locus that codes for the acetohydroxyacid synthase, which is the target site of these herbicides. The mechanism of resistance endowed by modifier genes has not been characterized and it could be related to non-target-site resistance. The objective of this study was to evaluate the role of cytochrome P450 monooxygenases (P450s) in Imisun resistance. The response to imazapyr herbicide in combination with P450s inhibitor malathion was evaluated in 2 Imisun PLANT PROTECTION - Article Contribution of non-target-site resistance in imidazolinone-resistant Imisun sunflower Gabriela Breccia 1 *, Mercedes Gil 1 , Tatiana Vega , Emiliano Altieri 2 , Mariano Bulos 2 , Liliana Picardi 1 , Graciela Nestares

Effect of Ahasl1-1 and Ahasl1-4 alleles on herbicide resistance and its associated dominance in sunflower: Effect of Ahasl1 alleles on herbicide resistance and its associated dominance

BACKGROUND Acetohydroxyacid synthase large subunit 1 (Ahasl1) is a multiallelic locus involved in herbicide resistance in sunflower. Ahasl1-1 and Ahasl1-4 alleles harbor different point mutations that lead to different amino acid substitutions (Ala205Val and Trp574Leu, respectively). The objectives of this work were to evaluate the effect of these alleles at the enzymatic and whole-plant levels, and to determine the dominance relationships for imazapyr and metsulfuron-methyl herbicides. RESULTS Resistant near-isogenic lines showed significantly lower specific AHAS activity than susceptible near-isoline. However, kinetic studies indicated that mutations did not change AHAS pyruvate affinity. Dose-response for six near-isolines carrying different combinations of Ahasl1-1 and Ahasl1-4 alleles and two herbicides (imazapyr and metsulfuron-methyl) were evaluated at whole-plant and enzymatic levels. Ahasl1-1 allele conferred moderate resistance to imazapyr and low resistance to metsulfuron-methyl. Conversely, Ahasl1-4 allele endowed high levels of resistance for both herbicides. Dominance of resistance at whole-plant level showed a semi-dominant behavior among the alleles for both herbicides. CONCLUSION Ahasl1-4 allele confers higher resistance levels than Ahasl1-1 when evaluated with imazapyr and metsulfuron-methyl. Dominance estimations suggested that both parental lines should carry a resistance trait when developing hybrids.