Target-site analysis

To identify whether any of the sampled populations were resistant to ACCase and ALS MOA as a result of target-site resistance, polymerase chain reaction (PCR) and Pyrosequencing were used to identify target-site mutations. Mutations at seven nucleotide positions, two ALS and five ACCase (as described in section 1.6.2 and 1.6.3) were studied. Before herbicide application (section 2.3.2), a 2cm section of leaf tissue was excised from eight plants per population. Each piece of leaf tissue was placed into a separate well of a 96-deep-well plate. To each well a stainless steel bead and 400µL of extraction buffer (100mM Tris(HCl) and 1 M KCl, pH 9.5) were added. Plates were shaken (30rpm for 10 minutes) using a Qiagen TissueLyser II, and centrifuged (4000rpm for 10 minutes) using a Sigma laboratory 4K15C centrifuge to extract the DNA. 100µl of supernatant was transferred into a new 96- well plate, from which a second plate containing 5µl supernatant and 250µl of Sigma-Aldrich W3500 sterile filtered tissue culture water was produced. The 100µl plate was stored at -80°C, while the 5µl plate was used as the PCR DNA template.

2.3.3.2 PCR Conditions for identifying ALS TSR mutations

To identify single nucleotide polymorphisms (SNP) at position Pro-197 of the ALS gene, a 140bp biotinylated fragment was amplified using forward and reverse

primers (forward: 5’-GTGCTACCAACCTCGTCTC-3’; reverse: 5’-

GGAGCGGGTGACCTCTACAAT-3’). A Master mix consisting of 10µl forward primer, 10µl reverse primer, 1030µl Sigma-Aldrich W3500 sterile filtered tissue culture water and 2500µl of Taq polymerase was produced. 5µl of DNA template and 20µl master mix was combined in a PCR plate (total volume = 25µl) using a

Beckman Coulter Biomek 3000 workstation. The plate was sealed and centrifuged using an Eppendorf 5810R centrifuge at 4000rpm for 1 minute. Amplification of the target region was carried out using an Eppendorf Mastercycler Gradient PCR machine set to denature at 94°C for 30s for 45 cycles, anneal at 60°C for 30s for 45 cycles, and elongate at 72°C for 40s for 45 cycles. PCR products were stored at 4°C until use.

To identify single nucleotide polymorphisms (SNP) at position Pro-574 of the ALS gene, a 401bp biotinylated fragment was amplified using forward and reverse

primers (forward: 5’-ATTCAGGAGTTGGCACTGATT-3’; reverse: 5’-

AGCTCTTGCCGAAGTTCTGAT-3’). A Master mix consisting of 10µl forward primer, 10µl reverse primer, 1030µl Sigma-Aldrich W3500 sterile filtered tissue culture water and 2500µl of Taq polymerase was produced. 5µl of DNA template and 20µl master mix was combined in a PCR plate (total volume = 25µl) using a Beckman Coulter Biomek 3000 workstation. The plate was sealed and centrifuged using an Eppendorf 5810R centrifuge at 4000rpm for 1 minute. Amplification of the target region was carried out using an Eppendorf Mastercycler Gradient PCR machine set to denature at 94°C for 30s for 45 cycles, anneal at 54.2°C for 30s for 45 cycles, and elongate at 72°C for 40s for 45 cycles. PCR products were stored at 4°C until use.

2.3.3.3 PCR Conditions for identifying ACCase TSR mutations

To identify single nucleotide polymorphisms (SNP) at position Ile-1781 of the ACCase gene, a 182bp biotinylated fragment was amplified using forward and reverse primers (forward: 5’-GCACACAAGATGCAGCTAGATAGT-3’; reverse:

5’-  TCCGATTCCAACAGTTCGT-3’). A Master mix consisting of 10µl forward primer, 10µl reverse primer, 1030µl Sigma-Aldrich W3500 sterile filtered tissue culture water and 2500µl of Taq polymerase was produced. 5µl of DNA template and 20µl master mix was combined in a PCR plate (total volume = 25µl) using a Beckman Coulter Biomek 3000 workstation. The plate was sealed and centrifuged using an Eppendorf 5810R centrifuge at 4000rpm for 1 minute. Amplification of the target region was carried out using an Eppendorf Mastercycler Gradient PCR machine set to denature at 94°C for 30s for 45 cycles, anneal at 54.0°C for 30s for 45 cycles, and elongate at 72°C for 40s for 45 cycles. PCR products were stored at 4°C until use.

To identify single nucleotide polymorphisms (SNP) at position identify Trp-2027, Ile-2041, Asp-2078, Gly-2096 of the ACCase gene, a 481bp biotinylated fragment

was amplified using forward and reverse primers (forward: 5’-

TCCTGTTGGTGTTATAGCTG-3’; reverse: 5’-GGATCAAGCCTACCCATGCA- 3’). A Master mix consisting of 10µl forward primer, 10µl reverse primer, 1030µl Sigma-Aldrich W3500 sterile filtered tissue culture water and 2500µl of Taq polymerase was produced. 5µl of DNA template and 20µl master mix was combined in a PCR plate (total volume = 25µl) using a Beckman Coulter Biomek 3000 workstation. The plate was sealed and centrifuged using an Eppendorf 5810R centrifuge at 4000rpm for 1 minute. Amplification of the target region was carried out using an Eppendorf Mastercycler Gradient PCR machine set to denature at 94°C for 30s for 45 cycles, anneal at 54.1°C for 30s for 45 cycles, and elongate at 72°C for 40s for 45 cycles. PCR products were stored at 4°C until use.

2.3.3.4 Pyrosequencing

The PCR product was split into two 12µl aliquots for cleaning and sequencing. 70µl of binding buffer and beads was added to each aliquot and the mixture was shaken for five minutes. 12µl of annealing buffer containing the relevant sequence primer

(Table 2.3)  were added to each well of a Pyrosequencing plate. The bead bound

DNA was washed using a Qiagen Pyromark Q96 workstation in 70% ethanol, sodium hydroxide (NaOH), and wash buffer before being transferred to the sequencing plate and dried at 80°C for two minutes. The Qiagen Pyromark Q96 MD pyrosequencer was set up so that the 10 nucleotides surrounding the single nucleotide polymorphism (SNP) mutation were sequenced. Peaks on the resulting pyrograms indicated the frequency of each nucleotide present.

Table 2.3: Primers used for pyrosequencing of ALS and ACCase target-site mutations.

Site of Action TSR mutation Sequencing Primer

ALS Pro-197 _{Trp-574 5’-CAACATCTGGGAATGGTGGTGCAG-3’} 5’-ATGGTCGCTATCACGGGACAGGTT-3’

ACCase Ile-1781 5’-ATGGACTAGGTGTGGAGAAC-3’ Trp-2027 5’-CCTCTGTTCATACTTGCTAAC-3’ Ile-2041 5’-GCAAAGAGATCTTTTTGAAGGA-3’ Asp-2078 5’-GTGGAGGAGCCTGGGTCGTGATT-3’ Gly-2096 5’-GCTATGCTGAGAGGACTGCAAAG-3’