Acetolactate synthase (ALS) inhibitors are widely used herbicides and their intense use in rice crops has resulted in many resistant weed populations. The polyploid species Echinochloa crus-galli (L.) Beauv. (barnyardgrass) is the most noxious weed that infests Italian rice felds. Previous experiments confrmed a broad cross-resistance pattern to ALS inhibitors in several populations of E. crus-galli collected in the north of Italy at both the whole-plant and enzyme activity levels. A target- site-mediated resistance mechanism was confrmed in fve resistant populations, where a G-T nucleotide substitution, giving a Trp-Leu change at amino acid 574, was detected. Further investigations were performed to characterise the molecular biology of ALS loci and alleles in this polyploid species. Southern blotting analysis was performed to detect the copy number of the ALS gene in the genome of E. crus-galli. Starting from the sequences obtained in the frst experiments, specifc primers were designed and three or four plants from each population were analysed. ALS clones were obtained from cDNA and 10 - 20 clone sequences were aligned and analysed through phylogenetic trees. A molecular marker, called AS-CAPS (Allele Specifc - Cleaved Amplifed Polymorphic Sequences), was designed and validated for the rapid detection of the mutation at codon 574. The Southern blotting analysis clearly showed the presence of multiple copies of the ALS gene. Phylogenetic analyses divided the cloned sequences into two clusters, ALS1 and ALS2. The mutation conferring the Trp-574-Leu substitution was detected in all resistant populations, and it was always found in ALS1, in homozygous or heterozygous status. The results of the AS-CAPS-574 marker analysis were in keeping with the sequencing results obtained. Further experiments using both allele-specifc and non-allele-specifc primers demonstrated the reliability of the molecular marker for the rapid detection of the 574 codon mutation in this hexaploid species.
Molecular evaluations on a polyploid species, Echinochloa crus-galli, resistant to ALS inhibitors
Panozzo S;Sattin M
2013
Abstract
Acetolactate synthase (ALS) inhibitors are widely used herbicides and their intense use in rice crops has resulted in many resistant weed populations. The polyploid species Echinochloa crus-galli (L.) Beauv. (barnyardgrass) is the most noxious weed that infests Italian rice felds. Previous experiments confrmed a broad cross-resistance pattern to ALS inhibitors in several populations of E. crus-galli collected in the north of Italy at both the whole-plant and enzyme activity levels. A target- site-mediated resistance mechanism was confrmed in fve resistant populations, where a G-T nucleotide substitution, giving a Trp-Leu change at amino acid 574, was detected. Further investigations were performed to characterise the molecular biology of ALS loci and alleles in this polyploid species. Southern blotting analysis was performed to detect the copy number of the ALS gene in the genome of E. crus-galli. Starting from the sequences obtained in the frst experiments, specifc primers were designed and three or four plants from each population were analysed. ALS clones were obtained from cDNA and 10 - 20 clone sequences were aligned and analysed through phylogenetic trees. A molecular marker, called AS-CAPS (Allele Specifc - Cleaved Amplifed Polymorphic Sequences), was designed and validated for the rapid detection of the mutation at codon 574. The Southern blotting analysis clearly showed the presence of multiple copies of the ALS gene. Phylogenetic analyses divided the cloned sequences into two clusters, ALS1 and ALS2. The mutation conferring the Trp-574-Leu substitution was detected in all resistant populations, and it was always found in ALS1, in homozygous or heterozygous status. The results of the AS-CAPS-574 marker analysis were in keeping with the sequencing results obtained. Further experiments using both allele-specifc and non-allele-specifc primers demonstrated the reliability of the molecular marker for the rapid detection of the 574 codon mutation in this hexaploid species.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
