Table 1.
dCAPS primers designed to detect target-enzyme mutation at position Pro-106 in EPSPS gene in Lolium perenne.
Table 2.
Nucleotide sequence in EPSPS gene in glyphosate-resistant (O) and glyphosate-susceptible (SP) populations of perennial ryegrass.
Changes to codons from the consensus sequence are highlighted.
Fig 1.
Digestion patterns of three restriction enzymes, MscI (A), Sau96I (B) and NlaIV (C) used for the dCAPS markers for detecting mutations at position Pro-106 in EPSPS gene in glyphosate-resistant population O (R) and glyphosate-susceptible population SP (S). S1 and R1 represent undigested samples showing the 216 bp dCAPS amplicon from an individual each of population S and O, respectively. S2 represents restriction enzyme-digested samples for the glyphosate susceptible individual yielding a single ~180 bp fragment. This individual was homozygous for the wild-type glyphosate sensitive allele of the EPSPS gene that harbors the C nucleotide at the codon 106 required for restriction enzyme cleavage in these dCAPS assays. The glyphosate-resistant individual (R2) had a 216 bp and ~180 bp fragment showing the presence of a mutation at codon 106 of the EPSPS gene for one allele as well as the wild-type sensitive allele, indicating this plant was heterozygous for these alleles. M denotes the DNA size standard lanes showing the 200 and 300 bp fragments of the 1 kb Plus size marker ladder (NEB, UK). The samples were resolved and visualized by electrophoresis in an agarose lithium borate buffer (2% w v-1) gel containing 0.5 μg ml-1 ethidium bromide.
Fig 2.
Sequence chromatogram showing codons 105 to 107 of a 300 bp PCR amplified section of the EPSPS gene of a perennial ryegrass individual from glyphosate resistant population O.
As both alleles in the individual were sequenced simultaneously the chromatogram revealed a heterozygous state at the first base of codon 106 (circled) identified as a pyrimidine (Y) comprising the wild type glyphosate sensitive C nucleotide overlapping with the glyphosate resistant T mutation at this position. This non-synonymous mutation indicated one allele in this glyphosate resistant individual had a Pro (CCA) to Ser (TCA) substitution at codon 106.
Fig 3.
Digestion patterns of the restriction enzyme, Sau96I used for the dCAPS markers for detecting mutations at codon 106 in the EPSPS gene in 18 individuals from a glyphosate-resistant Blenheim population.
The presence of the 216 bp dCAPS amplicon indicated a non-cleaved resistant allele of the EPSPS gene, whereas the ~180 bp cleaved amplicon showed presence of the glyphosate-sensitive allele. RS and RR represent digested samples for glyphosate-resistant individuals that were heterozygous and homozygous, for SNP mutations conferring resistance at codon 106 in the EPSPS gene. M denotes the DNA size standard lanes showing the 200 and 300 bp fragments of the 1 kb Plus size marker ladder (NEB, UK). The samples were resolved and visualized by electrophoresis in an agarose lithium borate buffer (2% w v-1) gel containing 0.5 μg ml-1 ethidium bromide.
Fig 4.
Sequence chromatogram showing codons 105 to 107 of a 300 bp PCR amplified section of the EPSPS gene of a perennial ryegrass individual from Blenheim population.
As there were two resistance alleles at position 106, the chromatogram revealed a homozygous state at the first base of codon 106 (circled) identified as the glyphosate resistant T mutation at this position. This non-synonymous mutation indicated both alleles in this glyphosate resistant individual had a Pro (CCA) to Ser (TCA) substitution at codon 106.