gDNA chromatograms of reference plants used for the development of AS-LAMP in Lolium spp. resistant to herbicides
Description
The data included in this repository were used for the set-up of Allele-Specific Loop-Mediated Isothermal Amplification (AS-LAMP) protocols for the detection of target-site resistance in Lolium spp. Two type of herbicide resistance, to acetolactate synthase (ALS) inhibitors and to Acetyl-CoA carboxylase (ACCase) inhibitors, and the most frequent point mutations detected responsible for herbicide resistance, 197, 376 and 574 in ALS gene and 1781, 2041 and 2078 in ACCase gene, were considered. The sequences of some samples previously characterized with specific genotypes were used for the design of the LAMP primers sets specific for an allelic variant using the Primer explorer V5 software (http://primerexplorer.jp/lampv5e/index.html). For each target mutation two sets of specific primers were designed, one primer set specific for the nucleotide which identifies the wild type (WT, responsible for the susceptible, S, phenotype) and one primer set specific for the nucleotide which identifies the mutated (MUT, responsible for the resistant, R, phenotype). The WT/MUT primer sets were designed using as input sequences generated during the genotyping of the twelve characterized Lolium spp. populations (Scarabel at al., Front Plant Sci 2020;11:1–12). The sequences are reported as .abi file to be able to distinguish even heterozygous samples at the specific mutation points. The code of each sequence identifies: the origin of the sample (IT = Italy, GR = Greece, DK = Denmark), the population and plant number (e.g. 595.4, 20.7, etc.), the status of sample (wt or mut) and the number of target mutation (e.g. 197, 376, etc.).
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Steps to reproduce
Total genomic DNA (gDNA) was extracted from 0.1 g leaf tissue using the CTAB method (Doyle and Doyle, 1987, Phytochem. Bull. 19, 11–15.). A region of the CT domain of the plastidic ACCase gene was amplified by PCR on gDNA using the primers acclr9 (5’-ATGGTAGCCTGGATCTTGGACATG-3’) and acclr6 (5’-GGAAGTGTCATGCAATTCAGCAA-3’) (Zhang and Powles, 2006, New Phytol. 172, 636–645). PCR amplifications were performed using GoTaq DNA Polymerase kit (Promega, United States) in a 25 µL mixture including 5 µL of 5 × Colorless GoTaq Flexi Buffer, dNTPs mix (0.2 mM each), MgCl2 (3 mM), forward and reverse primers (0.4 µM each), 0.125 µL GoTaq DNA Polymerase, and 25 ng of gDNA. The thermocycler program was as follows: 95 °C for 2 min; 45 cycles of 95 °C for 30 s, 57 °C for 30 s, 72 °C for 2 min; 72 °C for 5 min. PCR products were purified with NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel GmbH & Co., Germany) following the manufacturer’s instructions. Once purified, PCR Similarly, a fragment of the ALS gene was amplified from each DNA extracted with primers LOL_ALS_F (5’-CCGCAAGGGCGCCGACATCCTCGT-3’) and ALS_LOL_R (5’-CGAAATCCTGCCATCACCTTCCAT-3’). PCR amplifications were performed using GoTaq DNA Polymerase kit (Promega, United States) as described for ACCase gene. The thermocycler program was as follows: 95 °C for 2 min; 45 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 2 min; 72 °C for 5 min. PCR products were purified and sequenced as described for ACCase gene.