Evolution and Spread of Glyphosate Resistant Barnyard Grass (Echinochloa colona (L.) Link) By Thai Hoan Nguyen This thesis is submitted in fulfilment of the requirements for the degree of Doctor of Philosophy School of Agriculture, Food and Wine Faculty of Sciences The University of Adelaide Waite Campus March, 2015 Abbreviations ACCase: Acetyl-CoA carboxylase AFLP: Amplified fragment length polymorphism AGRF: Australian Genome Research Facility ALS: Acetolactate synthase EPSP: 5-enolpyruvylshikimate-3-phosphate synthase HAT: Hour after treatment LD 50 : Lethal dosage (dose required to control 50% of individuals in the population) LSD: Least significant different NSW: New South Wales PCR: Polymerase chain reaction QLD: Queensland R/S: Resistance/susceptibility RAPD: Randomly amplified polymorphic DNAs RFLP: Restriction fragment length polymorphism SA: South Australia SE: Standard error SSR: Simple sequence repeats VIC: Victoria WA: Western Australia i Table of Contents Abbreviations i Table of contents ii List of tables vii List of figures ix Abstract xi Declaration xiii Tables Published with Consent from Copyright Holders in this Thesis xiv Figures Published with Consent from Copyright Holders in this Thesis xiv Acknowledgements xv Chapter 1: General Introduction Chapter 2: Literature Review 2.1 Introduction 2.2 Echinochloa spp 2.2.1 Geographical distribution of Echinochloa spp in the world 2.2.2 Biology 2.2.3 Agronomical features 2.2.4 Problems caused by Echinochloa spp in Australia 2.2.5 Herbicide resistance in Echinochloa spp 10 2.2.6 Herbicide resistance in weed species in Australia 11 2.3 Causes of resistance evolution 13 2.3.1 Genetic mutations endow resistance 13 2.3.2 Initial frequency of resistant alleles 14 2.3.3 Selection pressure 14 2.3.4 Inheritance of resistance 15 2.3.5 Gene migration 15 ii 2.3.6 Fitness of resistant individuals 16 2.3.7 Characteristics of the seed bank 17 2.4 Glyphosate 17 2.4.1 Properties of glyphosate 17 2.4.2 Mode of action 18 2.4.3 Glyphosate resistant weeds 19 2.4.4 Mechanisms of glyphosate resistance 20 2.5 Molecular markers 22 2.5.1 Scientific basis of the use of molecular makers in determining the spread of resistance evolution in Echinochloa spp 22 2.5.2 DNA fingerprinting techniques 22 2.5.3 DNA sequencing 24 2.6 Conclusions 25 Literature cited 26 Chapter 3: Genetic Diversity of Glyphosate Resistant Junglerice (Echinochloa colona) in New South Wales and Queensland 34 Materials and methods 36 Plant material 36 Glyphosate dose response experiment 38 AFLP analysis 39 Results and discussion 41 Response to glyphosate 41 AFLP analysis 43 Genetic diversity across E colona populations 43 Genetic diversity within populations 45 Acknowledgements 51 Literature cited 51 iii Chapter 4: Temperature Influences the Level of Glyphosate Resistance in Barnyard Grass (Echinochloa colona) 56 Abstract 56 Introduction 57 Materials and methods 58 2.1 Plant material 58 2.2 Temperature response to glyphosate 58 2.3 Identifying target-site mutations 59 2.4 EPSPS gene relative copy number 61 2.5 Shikimate assay 62 2.6 Effect of temperature on absorption and translocation of glyphosate 63 Results 64 3.1 Temperature response experiments 64 3.2 Target-site mutations 66 3.3 EPSPS gene copy number 67 3.4 Effect of temperature on shikimate accumulation 67 3.5 14C-glyphosate absorption and translocation as affected by temperature 68 Discussion 70 4.1 Temperature influences glyphosate resistance 70 4.2 Target-site contributes to glyphosate resistance 71 4.3 14C-glyphosate absorption and translocation as affected by temperature 71 Conclusions 74 Acknowledgements 75 References 75 Chapter 5: Inheritance of Evolved Glyphosate Resistance in Barnyard Grass (Echinochloa colona) from Australia 80 Abstract 80 5.1 Introduction 81 iv 5.2 Materials and methods 82 5.2.1 Plant material 82 5.2.2 Gene flow between resistant and susceptible individuals 83 5.2.3 Hand crossing of resistant and susceptible individuals 85 5.2.4 Sequencing of F and F progenies 85 5.2.5 Shikimate accumulation 86 5.2.6 Segregation test 87 5.2.7 Response to glyphosate 88 5.2.8 EPSPS cDNA sequencing 88 5.3 Results and discussion 89 5.3.1 Plant growth of the parental populations 89 5.3.2 Gene flow frequency 91 5.3.3 Detecting EPSPS gene mutation in F and F progenies 93 5.3.4 Shikimate assay 94 5.3.5 Segregation test 95 5.3.6 Dose response to glyphosate of F progenies 97 5.3.7 EPSPS cDNA sequencing 98 5.4 Conclusions 100 Literature cited 101 Chapter 6: General Discussion 107 6.1 Discussion of results 107 6.2 Conclusions 113 6.3 Contributions to knowledge 114 6.4 Future research 114 Literature cited 115 Appendices 121 Appendix 1: Geographical sites of towns where are nearest to origins of 65 E colona populations used in this research (Chapter 3) 121 v Appendix 2: Response of eleven E colona populations to different glyphosate rates in the dose response experiment (Chapter 3) 122 Appendix 3: Distance matrix from AFLPs based on jaccard’s coefficient in comparison between populations collected across Queensland and New South Wales, Australia (Chapter 3) 123 Appendix 4: Distance matrix from AFLPs based on jaccard’s coefficient in comparison within populations collected in New South Wales, Australia (Chapter 3) 128 Appendix 5: Number and name indexes of E colona populations in Chapter and Appendices and 141 Appendix 6: Dendrogram of the partial sequence of the predicted amino acid at codon 106 in the EPSPS gene of the susceptible population (Echi S) and the resistant population (A533.1) (Chapter 4) 142 Appendix 7: The sections of the treated leaf, the non-treated leaves, the stem and the roots of E colona plants were cut at harvest time points of 12, 24, 48 and 72 hours after glyphosate application (Chapter 4) 143 Appendix 8: Response of E colona populations to glyphosate (240 g a.i ha-1) at two different temperature levels (20 and 30oC) at three weeks after glyphosate application (Chapter 4) 144 Appendix 9: The pair of resistant (A533.1) and susceptible (Echi S) E colona individuals at flowering, and two single spikes of each resistant and susceptible individual were bagged with glassine bags as controls before anthesis in gene flow experiments (Chapter 5) 145 Appendix 10: Survivors of E colona in gene flow frequency experiment at 30 days after spraying glyphosate (Chapter 5) 145 Appendix 11: Growth time, flower-head number, 100-seed weight and germinability of the resistant (A533.1) and the susceptible (Echi S) populations of E colona in gene flow experiments (Chapter 5) 146 Appendix 12: Number of E colona plants before and after spraying glyphosate in the gene flow frequency experiment (Chapter 5) 147 vi List of Tables Chapter Table 2.1 Documented herbicide resistance in weed species in Australia 12 Chapter Table Location and resistance phenotype of E colona populations used in this study with resistance that was determined by treating with glyphosate at 270 g a.e ha-1 37 Table AFLP adaptors and primers 40 Table The glyphosate resistance levels of ten E colona populations 42 Table Between-population genetic structure: fragment lengths, total number of fragments, number and percentage of polymorphic fragments produced by each primer set used to analyse the polymorphisms of one individual from each of 62 E colona populations 44 Table Within-population genetic structure: fragment lengths, total number of fragments, number and percentage of polymorphic fragments produced by each primer set used to analyse the polymorphisms of one individual from each of two glyphosate resistant E colona populations (63 and 64) and one susceptible population (65) 47 Chapter Table Locations are nearest to origins of E colona populations used in this study 58 Table The primers and probes used to identify the target-site mutation and determine the genomic copy number of EPSPS and ALS using quantitative real-time PCR 60 vii Table Glyphosate dose required to control 50% (LD 50 ) (g a.e ha-1) of susceptible and resistant E colona populations at 20oC and 30oC 66 Table Nucleotide and predicted amino acid sequence of EPSPS DNA isolated from a susceptible and ten resistant populations of E colona 67 Chapter Table Growth time (transplanting, flowering and harvest) of the resistant (A533.1) and the susceptible (Echi S) populations of E colona in the gene flow experiment 89 Table Flower head number, 100-seed weight and germinability of the resistant (A533.1) and the susceptible (Echi S) populations of E colona in the gene flow experiment 90 Table Survival of F progenies from four parental susceptible plants in the gene flow experiment to determine gene flow frequency between resistant (A533.1) and susceptible (Echi S) E colona plants 91 Table Segregation of the F progenies from selfed F survivors of E colona from the gene flow experiment after glyphosate treatment at of 240 g a.e ha-1 96 Table Mutation at position 106 of the cDNA EPSPS gene detected after cloning of resistant and susceptible plants, and F progenies of E colona 99 viii List of Figures Chapter Figure 2.1 Chemical structure of glyphosate 17 Figure 2.2 Activity of glyphosate on reaction catalysed by enzyme 5-enolpyruvylshikimate-3-phosphate synthase 19 Chapter Figure UPGMA phenogram of the genetic relationship between E colona populations collected across QLD and NSW, Australia 46 Figure UPGMA phenogram showing the genetic relationship within two resistant populations (63 and 64 in Table 3.1) and the susceptible population (65) of E colona collected from three separate fields in NSW, Australia 49 Chapter Figure Response of E colona populations to glyphosate at 20oC and 30oC 65 Figure Shikimic acid accumulation of leaf discs from two resistant (A533.1 and A818) and one susceptible (Echi S) E colona populations at different glyphosate concentrations at 20oC and 30oC 68 Figure 14C-glyphosate absorbed and translocated to plant sections of two resistant (A533.1 and A818) and one susceptible (Echi S) E colona populations at 20oC and 30oC at 12, 24, 48 and 72 hours after glyphosate application 69 ix (Appendix continued) Individual 18512.16 18512.17 18512.19 18512.20 18512.23 18512.24 18512.25 18512.26 18512.27 18512.28 158-12.29 0.653 0.642 0.643 0.667 0.707 0.645 0.695 0.667 0.647 0.638 158-12.2 0.594 0.596 0.612 0.686 0.634 0.653 0.619 0.562 0.604 0.592 158-12.30 0.548 0.549 0.626 0.621 0.635 0.642 0.573 0.606 0.576 0.576 158-12.3 0.636 0.600 0.675 0.636 0.673 0.642 0.673 0.673 0.619 0.623 158-12.4 0.600 0.574 0.655 0.625 0.638 0.644 0.637 0.625 0.557 0.598 158-12.7 0.706 0.598 0.721 0.720 0.707 0.701 0.695 0.707 0.686 0.707 158-12.8 0.573 0.588 0.642 0.625 0.651 0.631 0.624 0.598 0.543 0.584 158-12.9 0.531 0.606 0.648 0.573 0.644 0.594 0.600 0.630 0.559 0.543 185-12.11 0.583 0.636 0.649 0.620 0.670 0.639 0.632 0.645 0.605 0.553 185-12.12 0.620 0.622 0.650 0.699 0.633 0.581 0.587 0.604 0.602 0.511 185-12.13 0.625 0.654 0.629 0.676 0.689 0.683 0.663 0.689 0.607 0.624 185-12.14 0.602 0.664 0.686 0.672 0.695 0.655 0.673 0.684 0.633 0.587 185-12.15 0.629 0.618 0.592 0.667 0.667 0.620 0.640 0.667 0.624 0.627 185-12.16 0.000 0.628 0.657 0.667 0.694 0.630 0.607 0.593 0.606 0.578 0.000 0.646 0.698 0.596 0.618 0.593 0.580 0.533 0.641 0.000 0.697 0.670 0.717 0.656 0.613 0.637 0.697 0.000 0.695 0.674 0.696 0.681 0.673 0.695 0.000 0.688 0.696 0.607 0.604 0.681 0.000 0.659 0.659 0.639 0.598 0.000 0.571 0.632 0.571 0.000 0.604 0.622 0.000 0.633 185-12.17 185-12.19 185-12.20 185-12.23 185-12.24 185-12.25 185-12.26 185-12.27 185-12.28 0.000 133 (Appendix continued) Individual 18512.2 18512.30 18512.6 18512.8 18512.9 7812.10 7812.11 7812.12 7812.13 7812.15 78-12.1 0.600 0.592 0.581 0.567 0.489 0.451 0.516 0.500 0.571 0.488 185-12.1 0.467 0.570 0.559 0.505 0.409 0.457 0.485 0.416 0.474 0.440 158-12.1 0.489 0.564 0.552 0.525 0.444 0.405 0.441 0.435 0.478 0.405 78-12.18 0.755 0.804 0.781 0.805 0.760 0.770 0.759 0.755 0.765 0.817 185-12.21 0.610 0.602 0.606 0.617 0.634 0.587 0.614 0.623 0.544 0.600 78-12.5 0.600 0.617 0.583 0.571 0.600 0.563 0.528 0.587 0.505 0.577 78-12.21 0.728 0.700 0.720 0.726 0.698 0.755 0.676 0.703 0.650 0.755 158-12.25 0.740 0.700 0.766 0.780 0.734 0.693 0.767 0.728 0.714 0.706 158-12.6 0.567 0.652 0.694 0.655 0.623 0.542 0.627 0.596 0.584 0.557 185-12.10 0.519 0.580 0.630 0.547 0.495 0.509 0.518 0.519 0.562 0.550 185-12.18 0.628 0.631 0.622 0.608 0.626 0.571 0.590 0.613 0.615 0.571 185-12.22 0.579 0.656 0.672 0.565 0.580 0.515 0.610 0.618 0.582 0.570 185-12.29 0.616 0.607 0.584 0.515 0.602 0.562 0.607 0.602 0.546 0.592 185-12.3 0.485 0.568 0.596 0.532 0.547 0.505 0.528 0.500 0.534 0.442 185-12.4 0.515 0.569 0.544 0.546 0.534 0.490 0.542 0.545 0.549 0.550 185-12.5 0.549 0.571 0.547 0.550 0.538 0.495 0.559 0.577 0.566 0.510 185-12.7 0.556 0.578 0.539 0.514 0.571 0.560 0.510 0.510 0.500 0.574 78-12.14 0.515 0.650 0.619 0.558 0.546 0.475 0.554 0.544 0.574 0.490 158-12.5 0.735 0.730 0.784 0.754 0.729 0.713 0.752 0.723 0.670 0.725 158-12.10 0.500 0.598 0.560 0.575 0.505 0.420 0.544 0.500 0.536 0.473 158-12.11 0.545 0.605 0.596 0.559 0.547 0.442 0.541 0.515 0.548 0.505 158-12.12 0.605 0.610 0.669 0.579 0.557 0.519 0.538 0.568 0.583 0.545 158-12.13 0.565 0.631 0.636 0.588 0.603 0.500 0.548 0.538 0.568 0.582 158-12.14 0.567 0.602 0.632 0.593 0.525 0.526 0.548 0.537 0.598 0.542 158-12.15 0.596 0.589 0.606 0.606 0.648 0.542 0.562 0.582 0.584 0.557 158-12.16 0.573 0.594 0.625 0.673 0.629 0.562 0.581 0.602 0.604 0.592 158-12.17 0.515 0.582 0.558 0.546 0.589 0.520 0.514 0.560 0.505 0.550 158-12.18 0.657 0.657 0.624 0.648 0.654 0.619 0.657 0.615 0.588 0.619 158-12.19 0.578 0.611 0.664 0.626 0.631 0.596 0.598 0.578 0.553 0.596 158-12.20 0.533 0.600 0.590 0.577 0.580 0.538 0.559 0.548 0.521 0.553 158-12.21 0.557 0.593 0.636 0.609 0.545 0.561 0.566 0.557 0.574 0.531 158-12.22 0.565 0.642 0.577 0.592 0.582 0.600 0.629 0.625 0.583 0.570 158-12.23 0.633 0.623 0.641 0.676 0.576 0.594 0.648 0.673 0.577 0.579 158-12.24 0.613 0.682 0.636 0.648 0.640 0.617 0.657 0.642 0.629 0.632 158-12.26 0.612 0.591 0.594 0.582 0.624 0.531 0.524 0.510 0.515 0.545 158-12.27 0.551 0.661 0.604 0.616 0.607 0.584 0.600 0.536 0.510 0.584 158-12.28 158-12.29 0.653 0.583 0.655 0.617 0.608 0.594 0.633 0.607 0.638 0.598 0.616 0.543 0.604 0.617 0.626 0.612 0.557 0.586 0.630 0.616 134 (Appendix continued) Individual 18512.2 18512.30 18512.6 18512.8 18512.9 7812.10 7812.11 7812.12 7812.13 7812.15 158-12.2 0.540 0.626 0.606 0.617 0.570 0.485 0.550 0.540 0.571 0.559 158-12.30 0.537 0.627 0.564 0.579 0.610 0.526 0.490 0.537 0.556 0.571 158-12.3 0.533 0.581 0.584 0.522 0.509 0.495 0.491 0.519 0.537 0.524 158-12.4 0.505 0.558 0.573 0.561 0.550 0.449 0.518 0.520 0.524 0.495 158-12.7 0.717 0.667 0.709 0.716 0.712 0.695 0.702 0.717 0.627 0.708 158-12.8 0.575 0.607 0.586 0.561 0.550 0.510 0.545 0.520 0.565 0.465 158-12.9 0.505 0.573 0.534 0.495 0.538 0.479 0.476 0.457 0.464 0.510 185-12.11 0.560 0.530 0.607 0.522 0.586 0.550 0.543 0.476 0.495 0.602 185-12.12 0.580 0.533 0.590 0.591 0.553 0.541 0.547 0.566 0.525 0.598 185-12.13 0.587 0.568 0.634 0.609 0.519 0.505 0.629 0.515 0.615 0.535 185-12.14 0.591 0.574 0.612 0.577 0.669 0.595 0.538 0.579 0.569 0.642 185-12.15 0.563 0.596 0.587 0.536 0.524 0.525 0.559 0.549 0.593 0.539 185-12.16 0.598 0.630 0.580 0.594 0.638 0.602 0.577 0.489 0.586 0.616 185-12.17 0.570 0.670 0.610 0.596 0.600 0.589 0.578 0.615 0.602 0.604 185-12.19 0.630 0.658 0.612 0.624 0.615 0.647 0.645 0.644 0.618 0.620 185-12.20 0.667 0.679 0.660 0.619 0.689 0.629 0.654 0.680 0.626 0.670 185-12.23 0.639 0.679 0.673 0.670 0.663 0.629 0.654 0.706 0.640 0.629 185-12.24 0.602 0.648 0.598 0.598 0.630 0.621 0.635 0.632 0.604 0.663 185-12.25 0.667 0.654 0.646 0.657 0.650 0.642 0.600 0.562 0.533 0.613 185-12.26 0.611 0.654 0.686 0.657 0.596 0.615 0.588 0.625 0.583 0.570 185-12.27 0.580 0.625 0.617 0.604 0.581 0.541 0.547 0.621 0.569 0.556 185-12.28 0.639 0.602 0.562 0.606 0.663 0.570 0.602 0.581 0.583 0.585 185-12.2 0.000 0.631 0.583 0.515 0.545 0.500 0.538 0.511 0.560 0.531 0.000 0.626 0.613 0.629 0.557 0.586 0.579 0.542 0.583 0.000 0.555 0.596 0.559 0.537 0.610 0.571 0.587 0.000 0.559 0.561 0.527 0.557 0.505 0.547 0.000 0.505 0.555 0.587 0.562 0.549 0.000 0.529 0.531 0.535 0.422 0.000 0.495 0.500 0.515 0.000 0.452 0.484 0.000 0.490 185-12.30 185-12.6 185-12.8 185-12.9 78-12.10 78-12.11 78-12.12 78-12.13 78-12.15 0.000 135 (Appendix continued) Individual 7812.16 7812.17 7812.19 7812.20 7812.22 7812.23 7812.24 7812.25 7812.26 7812.27 78-12.1 0.571 0.573 0.488 0.506 0.536 0.556 0.553 0.539 0.506 0.613 185-12.1 0.641 0.667 0.582 0.538 0.562 0.581 0.562 0.592 0.505 0.615 158-12.1 0.607 0.591 0.544 0.458 0.523 0.541 0.506 0.588 0.477 0.583 78-12.18 0.809 0.835 0.788 0.781 0.773 0.798 0.798 0.800 0.758 0.775 185-12.21 0.617 0.657 0.642 0.657 0.625 0.630 0.570 0.611 0.573 0.620 78-12.5 0.635 0.673 0.606 0.697 0.654 0.686 0.629 0.675 0.604 0.707 78-12.21 0.663 0.755 0.697 0.752 0.745 0.644 0.653 0.702 0.693 0.724 158-12.25 0.722 0.742 0.723 0.714 0.707 0.729 0.733 0.726 0.706 0.724 158-12.6 0.648 0.713 0.657 0.604 0.667 0.673 0.640 0.651 0.586 0.673 185-12.10 0.670 0.713 0.616 0.593 0.587 0.655 0.613 0.647 0.537 0.655 185-12.18 0.605 0.737 0.620 0.622 0.615 0.605 0.600 0.683 0.646 0.653 185-12.22 0.692 0.735 0.649 0.573 0.594 0.676 0.645 0.689 0.609 0.640 185-12.29 0.609 0.720 0.622 0.625 0.646 0.680 0.619 0.670 0.660 0.716 185-12.3 0.635 0.660 0.592 0.551 0.615 0.592 0.574 0.615 0.563 0.585 185-12.4 0.638 0.636 0.551 0.505 0.561 0.622 0.618 0.590 0.505 0.559 185-12.5 0.667 0.689 0.623 0.598 0.606 0.567 0.578 0.631 0.553 0.615 185-12.7 0.620 0.673 0.546 0.592 0.614 0.604 0.614 0.651 0.560 0.648 78-12.14 0.646 0.706 0.590 0.592 0.573 0.618 0.613 0.637 0.602 0.634 158-12.5 0.785 0.776 0.755 0.695 0.727 0.763 0.765 0.769 0.713 0.755 158-12.10 0.629 0.613 0.568 0.506 0.579 0.598 0.579 0.608 0.521 0.590 158-12.11 0.663 0.647 0.592 0.566 0.588 0.647 0.629 0.575 0.549 0.661 158-12.12 0.676 0.684 0.611 0.574 0.632 0.707 0.643 0.675 0.609 0.683 158-12.13 0.650 0.636 0.609 0.611 0.606 0.648 0.643 0.605 0.569 0.661 158-12.14 0.633 0.632 0.511 0.574 0.583 0.617 0.505 0.584 0.557 0.608 158-12.15 0.648 0.687 0.588 0.574 0.583 0.632 0.598 0.598 0.571 0.608 158-12.16 0.593 0.624 0.636 0.565 0.589 0.562 0.604 0.561 0.577 0.600 158-12.17 0.609 0.663 0.566 0.596 0.561 0.622 0.546 0.655 0.550 0.651 158-12.18 0.624 0.681 0.635 0.609 0.632 0.622 0.632 0.683 0.619 0.653 158-12.19 0.628 0.692 0.625 0.586 0.566 0.612 0.648 0.670 0.623 0.655 158-12.20 0.616 0.645 0.570 0.571 0.565 0.568 0.611 0.582 0.553 0.660 158-12.21 0.591 0.677 0.592 0.548 0.588 0.591 0.573 0.629 0.546 0.612 158-12.22 0.619 0.633 0.602 0.589 0.613 0.602 0.567 0.568 0.600 0.636 158-12.23 0.628 0.641 0.611 0.567 0.606 0.656 0.606 0.647 0.636 0.616 158-12.24 0.588 0.696 0.649 0.607 0.630 0.652 0.600 0.643 0.660 0.653 158-12.26 0.649 0.660 0.644 0.606 0.600 0.589 0.600 0.573 0.588 0.673 158-12.27 0.674 0.709 0.627 0.602 0.582 0.585 0.537 0.648 0.625 0.619 158-12.28 158-12.29 0.605 0.636 0.663 0.743 0.619 0.574 0.649 0.635 0.629 0.643 0.604 0.663 0.643 0.643 0.654 0.654 0.616 0.657 0.610 0.610 136 (Appendix continued) Individual 7812.16 7812.17 7812.19 7812.20 7812.22 7812.23 7812.24 7812.25 7812.26 7812.27 158-12.2 0.673 0.630 0.588 0.561 0.584 0.630 0.584 0.585 0.545 0.607 158-12.30 0.586 0.673 0.573 0.574 0.583 0.602 0.553 0.598 0.600 0.648 158-12.3 0.647 0.682 0.579 0.539 0.589 0.645 0.602 0.638 0.565 0.647 158-12.4 0.636 0.660 0.581 0.525 0.590 0.608 0.577 0.551 0.538 0.625 158-12.7 0.652 0.730 0.646 0.716 0.709 0.717 0.670 0.704 0.683 0.650 158-12.8 0.650 0.673 0.607 0.569 0.590 0.594 0.590 0.616 0.593 0.625 158-12.9 0.628 0.653 0.584 0.557 0.566 0.612 0.536 0.594 0.569 0.630 185-12.11 0.682 0.702 0.616 0.618 0.600 0.679 0.613 0.658 0.589 0.667 185-12.12 0.615 0.696 0.600 0.588 0.596 0.629 0.610 0.610 0.612 0.682 185-12.13 0.649 0.722 0.657 0.580 0.615 0.660 0.602 0.652 0.590 0.624 185-12.14 0.685 0.681 0.644 0.669 0.641 0.736 0.664 0.693 0.630 0.680 185-12.15 0.667 0.676 0.610 0.570 0.645 0.663 0.632 0.618 0.553 0.664 185-12.16 0.667 0.704 0.589 0.649 0.600 0.634 0.600 0.667 0.602 0.637 185-12.17 0.624 0.637 0.576 0.624 0.646 0.607 0.617 0.588 0.574 0.640 185-12.19 0.670 0.745 0.676 0.667 0.633 0.667 0.673 0.730 0.660 0.667 185-12.20 0.725 0.745 0.673 0.649 0.642 0.691 0.642 0.729 0.670 0.676 185-12.23 0.635 0.648 0.660 0.573 0.670 0.618 0.598 0.640 0.629 0.622 185-12.24 0.610 0.685 0.609 0.626 0.649 0.670 0.663 0.604 0.621 0.683 185-12.25 0.617 0.678 0.630 0.663 0.626 0.648 0.611 0.581 0.567 0.663 185-12.26 0.619 0.663 0.571 0.620 0.582 0.602 0.628 0.626 0.600 0.594 185-12.27 0.584 0.643 0.600 0.588 0.582 0.600 0.650 0.636 0.570 0.645 185-12.28 0.619 0.677 0.617 0.649 0.642 0.602 0.613 0.640 0.629 0.650 185-12.2 0.637 0.677 0.562 0.594 0.511 0.591 0.573 0.602 0.576 0.651 185-12.30 0.626 0.735 0.585 0.639 0.620 0.676 0.620 0.619 0.634 0.664 185-12.6 0.632 0.670 0.602 0.644 0.676 0.644 0.598 0.649 0.613 0.645 185-12.8 0.683 0.703 0.627 0.617 0.611 0.667 0.598 0.669 0.625 0.711 185-12.9 0.635 0.647 0.592 0.580 0.602 0.620 0.574 0.627 0.604 0.661 78-12.10 0.626 0.611 0.552 0.522 0.547 0.611 0.532 0.550 0.521 0.588 78-12.11 0.612 0.688 0.544 0.613 0.607 0.598 0.594 0.632 0.570 0.664 78-12.12 0.622 0.690 0.577 0.608 0.602 0.621 0.558 0.602 0.561 0.638 78-12.13 0.638 0.676 0.536 0.610 0.531 0.594 0.576 0.642 0.550 0.639 78-12.15 0.641 0.667 0.610 0.568 0.592 0.549 0.562 0.619 0.580 0.602 78-12.16 0.000 0.631 0.565 0.600 0.625 0.647 0.625 0.609 0.580 0.634 0.000 0.684 0.615 0.680 0.674 0.667 0.622 0.596 0.673 0.000 0.629 0.579 0.567 0.548 0.621 0.596 0.631 0.000 0.517 0.615 0.596 0.637 0.553 0.606 0.000 0.578 0.589 0.670 0.562 0.627 0.000 0.578 0.608 0.596 0.604 0.000 0.590 0.562 0.641 0.000 0.520 0.626 0.000 0.574 78-12.17 78-12.19 78-12.20 78-12.22 78-12.23 78-12.24 78-12.25 78-12.26 0.000 78-12.27 137 (Appendix continued) Individual 7812.29 7812.2 7812.30 7812.3 7812.4 7812.6 7812.7 7812.8 7812.9 7812.28 78-12.1 0.534 0.429 0.630 0.516 0.536 0.535 0.585 0.489 0.511 0.500 185-12.1 0.515 0.436 0.604 0.500 0.446 0.398 0.505 0.474 0.366 0.548 158-12.1 0.522 0.402 0.643 0.473 0.495 0.471 0.542 0.478 0.418 0.506 78-12.18 0.750 0.781 0.849 0.798 0.789 0.804 0.760 0.817 0.802 0.779 185-12.21 0.633 0.667 0.648 0.616 0.661 0.636 0.658 0.609 0.611 0.600 78-12.5 0.544 0.515 0.676 0.542 0.518 0.530 0.561 0.444 0.537 0.618 78-12.21 0.673 0.731 0.752 0.716 0.744 0.677 0.722 0.710 0.732 0.698 158-12.25 0.699 0.720 0.752 0.704 0.744 0.716 0.722 0.734 0.697 0.684 158-12.6 0.621 0.550 0.676 0.617 0.640 0.567 0.583 0.623 0.571 0.683 185-12.10 0.532 0.477 0.669 0.531 0.496 0.461 0.561 0.481 0.444 0.575 185-12.18 0.667 0.650 0.628 0.634 0.657 0.628 0.653 0.667 0.654 0.633 185-12.22 0.604 0.577 0.712 0.600 0.612 0.618 0.629 0.617 0.607 0.661 185-12.29 0.557 0.526 0.683 0.596 0.570 0.602 0.574 0.545 0.590 0.621 185-12.3 0.585 0.515 0.664 0.582 0.545 0.573 0.561 0.547 0.466 0.618 185-12.4 0.587 0.515 0.679 0.500 0.559 0.588 0.575 0.534 0.510 0.532 185-12.5 0.575 0.519 0.667 0.560 0.549 0.520 0.591 0.495 0.514 0.608 185-12.7 0.540 0.594 0.676 0.618 0.568 0.556 0.571 0.571 0.574 0.527 78-12.14 0.596 0.569 0.611 0.568 0.581 0.529 0.598 0.598 0.536 0.629 158-12.5 0.789 0.714 0.806 0.745 0.739 0.771 0.717 0.786 0.716 0.770 158-12.10 0.484 0.500 0.660 0.559 0.547 0.467 0.550 0.520 0.480 0.565 158-12.11 0.571 0.500 0.651 0.514 0.505 0.500 0.587 0.519 0.495 0.604 158-12.12 0.603 0.565 0.686 0.576 0.465 0.514 0.581 0.569 0.496 0.646 158-12.13 0.602 0.588 0.664 0.586 0.563 0.551 0.603 0.579 0.491 0.620 158-12.14 0.551 0.520 0.637 0.563 0.551 0.521 0.596 0.554 0.485 0.585 158-12.15 0.594 0.606 0.624 0.655 0.640 0.637 0.648 0.583 0.636 0.554 158-12.16 0.654 0.584 0.630 0.623 0.622 0.573 0.615 0.602 0.549 0.591 158-12.17 0.530 0.515 0.602 0.570 0.559 0.515 0.548 0.505 0.524 0.547 158-12.18 0.598 0.637 0.683 0.648 0.594 0.585 0.600 0.586 0.654 0.604 158-12.19 0.617 0.548 0.657 0.613 0.600 0.564 0.579 0.606 0.569 0.637 158-12.20 0.577 0.516 0.650 0.588 0.589 0.564 0.551 0.566 0.554 0.582 158-12.21 0.584 0.540 0.654 0.539 0.607 0.495 0.559 0.573 0.534 0.619 158-12.22 0.594 0.532 0.639 0.531 0.642 0.596 0.624 0.637 0.584 0.552 158-12.23 0.602 0.571 0.673 0.612 0.585 0.574 0.576 0.576 0.619 0.593 158-12.24 0.625 0.579 0.613 0.620 0.670 0.628 0.640 0.626 0.654 0.618 158-12.26 0.596 0.539 0.612 0.606 0.580 0.570 0.585 0.571 0.561 0.602 158-12.27 0.564 0.617 0.648 0.640 0.638 0.608 0.633 0.607 0.583 0.538 158-12.28 0.582 0.660 0.640 0.619 0.643 0.626 0.664 0.612 0.664 0.587 138 (Appendix continued) Individual 7812.29 7812.2 7812.30 7812.3 7812.4 7812.6 7812.7 7812.8 7812.9 7812.28 158-12.29 0.537 0.648 0.653 0.632 0.631 0.612 0.651 0.612 0.639 0.587 158-12.2 0.510 0.552 0.648 0.524 0.603 0.540 0.570 0.557 0.505 0.600 158-12.30 0.521 0.619 0.610 0.590 0.604 0.582 0.610 0.569 0.558 0.489 158-12.3 0.519 0.476 0.672 0.558 0.482 0.519 0.495 0.523 0.514 0.549 158-12.4 0.519 0.490 0.627 0.559 0.495 0.505 0.550 0.564 0.486 0.564 158-12.7 0.689 0.697 0.705 0.694 0.701 0.680 0.625 0.712 0.722 0.713 158-12.8 0.561 0.519 0.687 0.571 0.522 0.548 0.481 0.577 0.500 0.606 158-12.9 0.490 0.490 0.632 0.475 0.509 0.441 0.538 0.433 0.485 0.505 185-12.11 0.584 0.607 0.658 0.593 0.582 0.546 0.586 0.561 0.576 0.589 185-12.12 0.564 0.549 0.635 0.575 0.589 0.551 0.645 0.553 0.543 0.626 185-12.13 0.624 0.596 0.639 0.569 0.619 0.573 0.613 0.613 0.550 0.644 185-12.14 0.578 0.539 0.661 0.563 0.553 0.591 0.626 0.543 0.534 0.582 185-12.15 0.562 0.547 0.679 0.519 0.509 0.520 0.538 0.495 0.486 0.536 185-12.16 0.610 0.635 0.640 0.606 0.643 0.612 0.612 0.612 0.613 0.587 185-12.17 0.568 0.582 0.670 0.521 0.645 0.585 0.600 0.627 0.629 0.557 185-12.19 0.667 0.664 0.683 0.636 0.692 0.644 0.654 0.642 0.642 0.663 185-12.20 0.663 0.634 0.680 0.682 0.642 0.706 0.637 0.650 0.664 0.660 185-12.23 0.689 0.647 0.667 0.631 0.690 0.667 0.663 0.689 0.651 0.688 185-12.24 0.585 0.626 0.700 0.610 0.636 0.632 0.657 0.602 0.631 0.591 185-12.25 0.621 0.646 0.680 0.657 0.691 0.653 0.676 0.608 0.637 0.614 185-12.26 0.622 0.634 0.625 0.604 0.702 0.625 0.676 0.650 0.570 0.645 185-12.27 0.564 0.563 0.697 0.602 0.661 0.566 0.594 0.607 0.609 0.640 185-12.28 0.594 0.577 0.611 0.604 0.630 0.611 0.650 0.610 0.625 0.600 185-12.2 0.556 0.554 0.614 0.567 0.582 0.511 0.573 0.573 0.520 0.589 185-12.30 0.628 0.602 0.655 0.600 0.588 0.593 0.580 0.568 0.518 0.610 185-12.6 0.539 0.593 0.685 0.578 0.591 0.569 0.609 0.543 0.586 0.571 185-12.8 0.556 0.568 0.670 0.566 0.504 0.583 0.571 0.545 0.586 0.573 185-12.9 0.571 0.570 0.688 0.542 0.595 0.545 0.600 0.533 0.537 0.618 78-12.10 0.531 0.500 0.631 0.544 0.546 0.546 0.535 0.520 0.480 0.564 78-12.11 0.495 0.523 0.643 0.550 0.513 0.524 0.514 0.514 0.477 0.540 78-12.12 0.541 0.525 0.614 0.581 0.556 0.444 0.545 0.559 0.520 0.543 78-12.13 0.485 0.515 0.642 0.557 0.545 0.515 0.575 0.490 0.524 0.516 78-12.15 0.545 0.515 0.618 0.529 0.573 0.531 0.563 0.563 0.495 0.548 78-12.16 0.620 0.660 0.652 0.615 0.704 0.575 0.621 0.635 0.636 0.628 78-12.17 0.686 0.657 0.716 0.692 0.722 0.703 0.722 0.673 0.685 0.656 78-12.19 0.516 0.588 0.634 0.573 0.613 0.562 0.592 0.578 0.594 0.517 78-12.20 0.660 0.531 0.636 0.615 0.589 0.622 0.621 0.608 0.569 0.613 139 (Appendix continued) Individual 7812.29 7812.2 7812.30 7812.3 7812.4 7812.6 7812.7 7812.8 7812.9 7812.28 78-12.22 0.614 0.570 0.602 0.610 0.634 0.616 0.642 0.602 0.590 0.591 78-12.23 0.633 0.588 0.635 0.614 0.664 0.591 0.673 0.660 0.608 0.596 78-12.24 0.586 0.526 0.588 0.596 0.609 0.573 0.574 0.602 0.590 0.576 78-12.25 0.626 0.636 0.667 0.583 0.699 0.629 0.675 0.615 0.640 0.606 78-12.26 0.574 0.573 0.670 0.571 0.635 0.604 0.590 0.577 0.552 0.579 78-12.27 0.623 0.658 0.664 0.643 0.675 0.651 0.684 0.684 0.649 0.644 78-12.29 0.000 0.553 0.676 0.538 0.593 0.526 0.571 0.485 0.547 0.461 0.000 0.636 0.480 0.514 0.495 0.543 0.515 0.476 0.557 0.000 0.620 0.678 0.654 0.676 0.676 0.652 0.660 0.000 0.590 0.539 0.595 0.528 0.519 0.526 0.000 0.471 0.491 0.505 0.425 0.585 0.000 0.559 0.453 0.475 0.574 0.000 0.547 0.537 0.590 0.000 0.509 0.561 0.000 0.578 78-12.2 78-12.30 78-12.3 78-12.4 78-12.6 78-12.7 78-12.8 78-12.9 78-12.28 0.000 140 Appendix 5: Number and name indexes of E colona populations in Chapter and Appendices and Population number Population name Population number Population name BYG2 34 RL13 BYG3 35 RL14 BYG4 36 RL2 BYG5 37 RL3 BYG6 38 RL4 Echi R 39 RL6 1301 40 RL8 182-12 41 Echi S RL10 42 RL16 10 RL15 43 140.1-12 11 RL19 44 1264.1 12 RL21 45 L594 13 RL22 46 967.1 14 A498.1 47 967.2 15 RL17 48 1264.2 16 1313.1 49 1307.1 17 A475 50 1307.2 18 1265.1 51 1307.3 19 1352.2 52 A491 20 1352.7 53 A516 21 163-12 54 A671.1 22 A1076.1 55 L593 23 A533.1 56 1352.3 24 A533.2 57 1352.4 25 A533.3 58 1352.5 26 A818 59 QBG4 27 RL18 60 1352.8 28 RL20 61 A492.2 29 RL24 62 A508 30 RL9 63 185-12 31 1352.1 64 78-12 32 RL1 65 158-12 33 RL11 141 Appendix 6: Dendrogram of the partial sequence of the predicted amino acid at codon 106 in the EPSPS gene of the susceptible population (Echi S - top) and the resistant population (A533.1 - bottom) showing the mutation (substitution of T for C leading to substitution of serine for proline) in the resistant population A533.1 (Chapter 4) 142 Appendix 7: The sections of the treated leaf, the non-treated leaves, the stem and the roots of E colona plants were cut at harvest time points of 12, 24, 48 and 72 hours after glyphosate application (Chapter 4) 143 Appendix 8: Response of E colona populations to glyphosate (240 g a.i ha-1) at two different temperature levels (20oC on the left and 30oC on the right) at three weeks after glyphosate application The order of photos from the left: Echi S (susceptible), 1307.3 (standard resistant) and A533.1 (most resistant) (Chapter 4) Experiment Experiment 144 Appendix 9: The pair of resistant (A533.1) and susceptible (Echi S) E colona individuals at flowering (left), and two single spikes of each resistant and susceptible individual were bagged with glassine bags as controls before anthesis (right) in gene flow experiments Letters inside the photos: S: susceptible plant, R: resistant plant (Chapter 5) Appendix 10: Survivors of E colona in gene flow frequency experiment at 30 days after spraying glyphosate (Chapter 5) Control (Echi S) Control (A533.1) F progenies of Echi S plants 145 Appendix 11: Growth time, flower-head number, 100-seed weight and germinability of the resistant (A533.1) and the susceptible (Echi S) populations of E colona in gene flow experiments (Chapter 5) Putting seeds on agar: Transplanting: Finish harvesting: Recording 100-seed weight: Recording germinability: 02/12/2011 12/12/2011 30/04/2012 06-07/06/2012 18-19/03/2013 Flowering Harvesting Start *Day FH Start *Day 100-seed weight (g) A533.1-1 16/01/12 35 183 13/02/12 63 0.11 85 A533.1-2 16/01/12 35 260 12/02/12 62 0.10 71 A533.1-3 16/01/12 35 133 14/02/12 64 0.10 48 A533.1-4 16/01/12 35 221 13/02/12 63 0.11 68 A533.1-5 18/01/12 37 229 18/02/12 68 0.10 75 A533.1-6 16/01/12 35 167 14/02/12 64 0.10 44 A533.1-7 16/01/12 35 238 16/02/12 66 0.10 86 A533.1-8 16/01/12 35 205 18/02/12 68 0.11 71 A533.1-9 17/01/12 36 231 17/02/12 67 0.10 29 A533.1-10 18/01/12 37 163 18/02/12 68 0.10 50 0.10 62.7 Individual Average 203.0 Germination (%) Echi S-1 16/01/12 35 267 13/02/12 63 0.12 96 Echi S-2 16/01/12 35 321 13/02/12 63 0.12 91 Echi S-3 16/01/12 35 193 14/02/12 64 0.11 88 Echi S-4 16/01/12 35 284 13/02/12 63 0.12 82 Echi S-5 16/01/12 35 312 13/02/12 63 0.11 63 Echi S-6 16/01/12 35 304 14/02/12 64 0.12 93 Echi S-7 16/01/12 35 294 16/02/12 66 0.11 86 Echi S-8 16/01/12 35 298 18/02/12 68 0.12 35 Echi S-9 16/01/12 35 307 13/02/12 63 0.12 93 Echi S-10 16/01/12 35 284 17/02/12 67 0.11 82 0.12 80.9 Average 286.4 * Number of days was counted since transplanting FH: Flower-head number 146 Appendix 12: Number of E colona plants before and after spraying glyphosate in the gene flow frequency experiment (Chapter 5) Time points of experiment: Putting seed on agar: Transplanting into trays: Spraying glyphosate: Recording survival: 18/01/2013 29/01/2013 28/02/2013 30/03/2013 Before spraying (2024 plants) After spraying (28 plants) Tray Echi S Echi S Echi S Echi S Echi S Echi S Echi S Echi S 17 47 48 49 1 10 43 50 49 0 1 24 31 45 45 0 13 47 49 45 3 42 49 46 1 49 44 48 1 46 42 50 0 45 40 49 0 42 39 46 0 10 49 40 48 1 11 44 47 48 0 12 29 18 47 1 13 31 44 0 14 50 0 15 45 16 47 17 31 18 44 19 31 20 29 Total Echi S (control) A533.1 (control) 68 554 511 891 12 24 25 24 23 0 0 20 18 23 19 20 18 23 19 Echi S 1, Echi S 2, Echi S 3, Echi S 4: E colona plants of which seeds were harvested from susceptible plants in Gene Flow experiment Glyphosate rate was applied: 240 g a.i ha-1 Survival rate after spraying glyphosate: 1.38% Echi S (control): 96 plants before spraying, A533.1 (control): 80 plants before and after spraying 147 [...]... barnyard grass (Echinochloa crus-galli)', Weed Science 46 ( 2), 163-169 Powles, S.B and Preston, C (200 6) 'Evolved glyphosate resistance in plants: Biochemical and genetic basis of resistance', Weed Technology 20 ( 2), 282-289 Storrie, A., Cook, T., Boutsalis, P., Penberthy, D and Moylan, P (200 8) Glyphosate resistance in awnless barnyard grass (Echinochloa colona (L. ) Link) and its implications for Australian... J.P and Gauvrit, C (200 7) 'Mechanisms of resistance to glyphosate in a ryegrass (Lolium multiflorum) biotype from Chile', Weed Science 55 ( 5), 435-440 Mooney, H.A and Hobbs, R.J (200 0) Invasive species in a changing world Island Press, Washington, D.C., US Norsworthy, J.K., Talbert, R.E and Hoagland, R.E (199 8) 'Chlorophyll fluorescence for rapid detection of propanil -resistant barnyard grass (Echinochloa. .. after glyphosate treatment of the resistant (A533. 1) and the susceptible (Echi S) populations of E colona in the rate test 90 Figure 2 The morphology of E colona flowers at the opening of the flower and before pollination, with pollen grains adhering to the stigmata 92 Figure 3 Shikimate accumulation of parental plants (A533.1 and Echi S) and the F 1 cross of E colona 95 Figure 4 Glyphosate. .. Argentina in 2009, QLD and WA (Australia) in 2009 and 2010 respectively (Heap, 201 4) This review will cover aspects of biological and agronomical characteristics of Echinochloa spp., properties and action mode of glyphosate, glyphosate resistance and causes, mechanisms of resistance in plant species, and molecular markers involving the studies of resistance evolution Relevant methodologies of the studies will... and Argentina (Heap, 201 4) Among these countries, Australia has been discovered to have the largest area of glyphosate resistant E colona with three states namely New South Wales (NSW), Queensland (QLD) and Western Australia (WA) (Heap, 201 4) Glyphosate resistance has occurred in many plant species (Lorraine-Colwill et al., 2003; Powles and Preston, 2006; Michitte et al., 2007; Gaines et al., 201 0). .. However, glyphosate resistance in E colona, was only recently discovered and is poorly understood (Storrie et al., 2008; Heap, 201 4) With the aim of improving understanding of glyphosate resistance evolution in E colona, this research was implemented to evaluate the genetic variability, assess the impacts of temperatures on the resistance level, and determine the inheritance and spread of glyphosate. .. all over the word and particularly in Australia owing to inhibiting the growth and losing the yield of crops (Holm et al., 1977; McGillion and Storrie, 200 6) These grass species have been commonly found in paddy fields around the world (Mooney and Hobbs, 200 0), in summer fallows and the grain-growing lands in QLD and NSW (Australia) (McGillion and Storrie, 200 6) Lands with moist soil and mild weather... potential spread of glyphosate resistant E Colona (Chapter 3) The response of glyphosate resistance in E colona to two different temperatures was also assessed and the reasons causing different responses were elucidated (Chapter 4) Finally, movement of the glyphosate resistance gene in E colona over the susceptible population and the pattern of the resistance inheritance were evaluated (Chapter 5) Overall,... resistance mechanisms, inheritance and spread of resistance were undertaken to better understand the evolution of glyphosate resistance in this species A survey of 65 barnyard grass populations collected from Queensland and New South Wales determined 34 populations were resistant to glyphosate with resistance levels ranging from 2 to 11-fold High genetic diversity within and between these populations was... Glyphosate dose response of susceptible and resistant populations of E colona and the F 2 population 98 x Abstract Echinochloa colona is an important summer-growing weed species in northern Australian cropping regions The intensive use of glyphosate in summer fallow operations has led to the appearance of glyphosate resistant E colona populations at a large number of sites Studies of the genetic diversity,