Sample and material
Echinochloa colona weed samples were collected from infested areas of the states of Queensland and New South Wales, Australia. These samples were purified and stored in the at the Queensland Alliance for Agriculture and Food Innovation (QAAFI), The University of Queensland, Gatton Campus. For DNA extraction well-matured seeds from each accession were selected and raised in pots in controlled conditions maintained at 30/20oC (day/night temperature). The plants were raised to 3–4 leaf stage for extraction of DNA. The seed samples of Echinochloa colona were stored in zip-lock bags at 4oC. The DNeasy Plant Mini Kit was purchased from Qiagen, Valencia, CA, USA. The DNA ladder and PCR reagents were purchased from Promega, Ltd., Madison, Wisconsin, USA. Primers were synthesized from Integrated DNA Technologies (IDT), Ltd., Coralville, Iowa, USA.
Solution and reagents used
Extraction buffer was 2.5% cetyl trimethyl ammonium bromide (CTAB), 100 mM Tris HCl (pH 8.0), 1.5 M NaCl, 25 mM ethylenediamine tetraaceticacid (EDTA), 3% polyvinylpolypyrrolidone (PVPP). Isopropanol 70% ethanol (v/v); Phenol: Chloroform: Isoamyl alcohol (25: 24: 1, v/v); Chloroform: Isoamyl alcohol (24:1, v/v); 0.3 M sodium acetate (pH 5.4); TE (Tris/EDTA) buffer: 10 mM Tris-HCl (pH 8.0); and 1 mM EDTA (pH 7.4). Commercially available kit (DNeasy Plant Mini Kit, Qiagen, Valencia, CA, USA). Taq DNA polymerase, Taq DNA polymerase buffer, RNase, Nucleotides dNTPs (G, A, T, C), SSR primers, 20 mL 0.5 M EDTA, Agarose gel and Ethidium bromide.
Equipment
The equipment utilized was a grinder or pestle mortar; multichannel pipettes (Eppendorf AG, Hamburg, Germany); plate mixer; plate centrifuge; water bath or oven or heating block, any type that can maintain the temperature at 65 °C; microcentrifuge; benchtop centrifuges with rotors if greater quantity of DNA is required; NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts,USA); polymerase chain reaction (PCR); electrophoresis systems; and Gel Documentation System (Gel DocTM EZ BIO-RAD Laboratories, Inc., Hercules, California, USA).
DNA extraction of E. colona leaves and seeds using Qiagen kit method
Echinochloa colona DNA from plant leaves and dry seed samples was extracted using the DNeasy Plant Mini Kit. The plants were raised to 3–4 leaf stage for extraction of DNA. Harvested leaves were placed in glassine bags and stored in ice. About 100 mg of leaves were ground to a fine powder using a pre-chilled pestle and mortar after adding liquid nitrogen to make the leaves brittle as well as to stop DNAase and other enzyme activities. For DNA extraction from dry seed, 20 mg of seeds were ground to a fine powder in pestle and mortar in liquid nitrogen. Further, the manufacturer’s instructions were followed for DNA from both the samples of leaves and dry seeds.
DNA extraction of barnyard leaves and seeds using CTAB method
The plants were raised to 3–4 leaf stage for extraction of DNA. Harvested leaves were placed in glassine bags and stored in ice. About 100 mg of leaves were ground to a fine powder using pre-chilled pestle and mortar after adding liquid nitrogen to make leaves brittle as well as to stop DNAase and other enzyme activities. Similarly, 20 mg of dry seeds were grounded to a fine powder in pestle and mortar in liquid nitrogen for DNA extraction from dry seeds. The powdered material of leaves, as well as seeds, were transferred to 1 ml of pre-warmed extraction buffer (CTAB buffer) and incubated for 60 minutes with occasional stirring. An equal volume of chloroform: isoamyl alcohol (24:1) was added in both the samples and slowly mixed by inverting the tubes for 10 minutes till it made a dark green emulsion. Both the samples were centrifuged for 10 minutes at 8000 rpm. The supernatant of both leaves, as well as seeds, were then transferred to another tube and again treated with chloroform: isoamyl alcohol (24:1), mixed slowly for 10 minutes and centrifugation was done. To the supernatant, 0.6 volume (400 µl) of ice-cold isopropanol was added and stored at 4 °C for 1–2 hours. Centrifugation was done at 13000 rpm for 10 minutes at 4 °C. The supernatant was discarded and the pellets were washed with 70% ethanol (200 µl – 300 µl) to remove contamination and were then air-dried. DNA was dissolved in 300 µl TE Buffer and stored at 40 °C. For purification of DNA, 300 µl of RNase (10 mg/ml) was added to the samples and incubated for 1 hour at 37 °C in a water bath. An equal volume of phenol:chloroform:isoamyl alcohol (PCI) (25:24:1) was added in both the samples, tilted for 10 minutes and centrifuged at 13,000 rpm for 10 minutes. The supernatant was collected in another tube, 0.6 volume of chilled isopropanol was added and centrifugation was done to get a pellet. DNA pellets of both samples were washed with 70% ethanol, air dried, and dissolved in TE (Tris-Cl, EDTA) buffer.
Quantitative and qualitative analysis of DNA
The quality of the isolated DNA was checked on 1% agarose gel. The concentration and relative purity of the isolated DNA were checked using Nanodrop ND-1000 (Agricultural Genomic Laboratory). DNA samples were diluted with distilled water and adjusted to 30 ng µL-1 [16]. The DNA extracted samples from leaves and seeds were stored at -20 °C until further use.
PCR amplification
PCR amplification was performed in a 25 µL reaction mixture that contained approximately 2 µL of 30 ng genomic DNA, 0.5 µL (50 pmol) of each primer (Integrated DNA technologies, Coralville, Iowa, USA), 12.5 µL PCR master mix (Promega, USA), 400 µM of dNTP mix, 0.5 µL of 2.5 U/µL Taq DNA polymerase (Promega, USA), 2.5 µL of 3.0 mM MgCl2 and nuclease-free H2O. Amplification was performed with a thermal cycler (Eppendorf Germany). The primer of gene EPSPS encoding 5-enolpyruvoylshikimate-3-phosphate (EPSP) synthase, a key enzyme in the shikimic acid pathway [17, 18]. An approximately 500 bp fragment of the EPSPS gene primers was amplified in an automated DNA thermal cycler with the cycle parameters as follows: 3 min denaturing at 94 °C; 40 cycles of 30 s denaturation at 94 °C, 30 s annealing at 56 °C and 1 min elongation at 68 °C, and a final extension for 7 min at 68 °C. PCR amplified products of all the primers were subjected to gel electrophoresis using 2.0% agarose gel in 1X TAE buffer at 100 V. The microsatellite markers were well resolved on 2% agarose gel with 3 hour run. The fragment sizes were determined by comparing with a 100 bp DNA ladder (Promega, USA) and the ethidium bromide stained gels were documented using Gel Documentation System (Gel DocTM EZ BIO-RAD, USA).