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Design and optimization of degenerated universal primers for the cloning of the plant acetolactate synthase conserved domains

Published online by Cambridge University Press:  20 January 2017

Mario Duran Prado ,
Rafael De Prado  and
Antonio R. Franco
Mario Duran Prado
Affiliation:
Bioquímica y Biología Molecular, Universidad de Córdoba, Campus de Rabanales, Edificio Severo Ochoa, Córdoba 14071, Spain
Rafael De Prado
Affiliation:
Química Agrícola y Edafología, Universidad de Córdoba, Campus de Rabanales, Edificio Marie Curie, Córdoba 14071, Spain
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Abstract

A set of universal and degenerate primers has been designed to clone (by polymerase chain reaction [PCR]) the conserved domains of the acetolactate synthase (ALS) gene where mutations confer resistance to ALS herbicides in plants. These primers were successful in cloning conserved domains of ALS in all monocotyledonous and dicotyledonous plants tested to date, as well as that of bacteria. Total genomic DNA was used as the source of target DNA because no introns were found in the sequences to be amplified. The design of the universal primers was performed after subtle modifications of the consensus degenerate hybrid oligonucleotide primers approach, which implies the synthesis of hybrid oligonucleotide primers containing fixed clamp 5′ and degenerate core 3′ sequences. Optimizations of PCR reactions were done according to a Taguchy approach described for the first time with degenerate oligonucleotides. This method optimizes a PCR reaction using four variables (deoxynucleoside triphosphate, DNA, primers, and Mg2+) under three different concentrations per variable using just nine reactions. The ALS herbicide-binding domains from many susceptible and resistant plants can be cloned and sequenced in a few hours by using only 100 mg of starting plant material, like one leaf or several small seedlings or seeds.

Keywords

Cetyltrimethylammonium bromidepigweed quitensis, Amaranthus quitensishairy beggarticks, Bidens pilosacorn poppy, Papaver rhoeascommon waterplantain, Alisma plantago-aquaticarigid ryegrass, Lolium rigidumALS resistanceprimer designPCRherbicide resistanceRAPD
Type
Physiology, Chemistry, and Biochemistry
Information
Weed Science , Volume 52 , Issue 4 , August 2004 , pp. 487 - 491
Copyright
Copyright © Weed Science Society of America 

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References

Literature Cited

Benjamin, D. C. and Clarkson, J. M. 1994. A simple procedure for optimising the polymerase chain reaction (PCR) using modified Taguchi methods. Nucleic Acid Res 22:3801–3805.Google Scholar
Boutsalis, P., Karotam, J., and Powles, S. B. 1999. Molecular basis of resistance to acetolactate synthase-inhibiting herbicides in Sisymbrium orientale and Brassica tournefortii . Pest. Sci 55:507–516.3.0.CO;2-G>CrossRefGoogle Scholar
Devereux, J., Haebarli, P., and Smithies, O. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acid Res 12:387–395.CrossRefGoogle ScholarPubMed
Devine, M. D. and Shukla, A. 2001. Altered target sites as a mechanism of herbicide resistance. Crop Prot 19:881–889.CrossRefGoogle Scholar
Gressel, J. 2002. Molecular Biology of Weed Control. London: Taylor & Francis. Pp. 155–160, 262–266.CrossRefGoogle Scholar
Hall, T. A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids. Symp. Ser 41:95–98.Google Scholar
Heap, I. 2004. International survey of herbicide resistant weeds. http://www.weedscience.org.Google Scholar
Mallory-Smith, C. A. D., Thill, D. C., and Dial, M. J. 1990. Identification of sulfonylurea herbicide-resistant prickly lettuce (Lactuca serriola). Weed Technol 4:163–168.Google Scholar
Rose, T. M., Schultz, E. R., Henikoff, J. G., Pietrovski, S., McCallum, C. M., and Henikoff, S. 1998. Consensus—degenerate hybrid oligonucleotide primers for amplification of distantly related sequences. Nucleic Acid Res 26:1628–1635.Google Scholar
Rychlik, W. and Rhoads, R. E. 1989. A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA. Nucleic Acids Res 17:8543–8551.CrossRefGoogle ScholarPubMed
Salas, M. 2001. Resistencia a herbicidas. Detección en campo y laboratorio. Pages 251–261 in De Prado, R. and Jorrín, J. eds. Uso de Herbicidas en la Agricultura del Siglo XXI. Córdoba, Spain: Servicio de Publicaciones de la Universidad de Córdoba.Google Scholar
Sibony, M., Michel, A., Haas, H. U., Rubin, B., and Hurle, K. 2001. Sulfometuron-resistant Amaranthus retroflexus: cross-resistance and molecular basis for resistance to acetolactate synthase (ALS) inhibiting herbicides. Weed Res 41:509–522.Google Scholar
Tranel, P. J. and Wright, T. R. 2002. Resistance of weeds to ALS-inhibiting herbicides: what have we learned? Weed Sci 50:700–712.Google Scholar
Wang, H., Qi, M., and Cutler, A. J. 1993. A simple method of preparing plant samples for PCR. Nucleic Acids Res 21:4153–4154.CrossRefGoogle ScholarPubMed
Wright, T. R., Bascomb, N. F., Sturner, S. F., and Penner, D. 1998. Biochemical mechanism and molecular basis for ALS-inhibiting herbicide resistance in sugarbeet (Beta vulgaris) somatic cell selections. Weed Sci 46:13–23.Google Scholar