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Can DNA Barcoding and Next Generation Sequencing (NGS) Technology Detect Invasive Plant Species in Seed Lots? Steve Jones and Marie-José Côté

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Can DNA Barcoding and Next Generation Sequencing (NGS) Technology Detect Invasive Plant Species in Seed Lots? Steve Jones and Marie-José Côté Can DNA barcoding and Next Generation Sequencing (NGS) technology detect invasive plant species in seed lots? Steve Jones and Marie-José Côté © 2017 Her Majesty the Queen in Right of Canada (Canadian Food Inspection Agency), all rights reserved. Use without permission is prohibited. Presentation overview Background: Species id and molecular testing What we did What we found Further research Future use Background Morphological id (dichotomous key) 1689: Richard Waller drawings and watercolours 1778: Jean-Baptiste Lamarck Flora Française text based Seed testing (correct crop species and other seed id) 1869: Prof. Dr. Friederick Nobbe first seed testing lab 1896: 119 seed labs worldwide 19 countries Molecular testing (DNA bar coding) 1953: DNA structure discovered: AG CT base pairs 1970s: Sanger sequencing 2019: Molecular species id in seed lots: let’s see…. Sanger sequencer DNA barcoding – how does it works? Top 5 lines: Amaranthus tuberculatus Bottom 3 lines: Amaranthus palmeri Herbarium specimens are subsampled for DNA extraction. DNA is then sequenced using universal DNA barcoding primers. DNA barcode sequences for ITS2, trnH-psbA are collected DNA barcoding – identifies differences C C A G A C C A T G C T C C C C C A T A G G G A C A .Asteraceae Centaurea stoebe QFA 511500 C C A G A C C A T G C T C C C C C A T A G G G A C A .Asteraceae Centaurea stoebe ALTA 118589 C T A G A C C A T G C T C C C T T A G A G G G A C G .Asteraceae Centaurea phrygia DAO 618362 C T A G A C C A T G C T C C C T T A G A G G G A C G .Asteraceae Centaurea phrygia DAO 854486 C C A A A C C A A G C T C C C C C A T A G G G A C G .Asteraceae Centaurea sulphurea DAO 871922 C C A A A C C A A G C T C C C C C A T A G G G A C G .Asteraceae Centaurea sulphurea US 1221543 C C A G A C C A T A C T C C C C C A T A G G G A T G .Asteraceae Centaurea iberica DAO 854476 C C A G A C C A T A C T C C C C C A T A G G G A T G .Asteraceae Centaurea iberica DAO 854478 C C A G A C C A T A C T T C C C C A T A G G G A T G .Asteraceae Centaurea calcitrapa DAO 870915 C C A G A C C A T A C T T C C C C A T A G G G A T G .Asteraceae Centaurea calcitrapa DAO 634363 C C A G A C C A T C C T C C C C C A T A G G G A C G .Asteraceae Centaurea solstitialis DAO 854503 C C A G A C C A T C C T C C C C C A T A G G G A C G .Asteraceae Centaurea solstitialis DAO 284465 C C A G A C C A T G C T C C C T C A T T G G G A T G .Asteraceae Centaurea sicula (nicaeensis) CAN 219416 C C A G A C C A T G C T C C C T C A T T G G G A T G .Asteraceae Centaurea sicula (nicaeensis) US 1667075 C C A A T C C T T G C T C C C T C A T A G G G A T G .Asteraceae Centaurea melitensis CAN 550565 C C A A T C C T T G C T C C C T C A T A G G G A T G .Asteraceae Centaurea melitensis CAN 208260 DNA barcoding can create phylogenic trees A T G C A T T A T G T - - C T C C C C T C T C G T G T G T G G A G T G - G G A A T A G A T C C T G G C C T C C T G G G C C C T T C C T Convolvulaceae Cuscuta campestris A T G C A T T A T G T - - C T C C C C T C T C G T G T G T G G A G T G - G G A A T A G A T C C T G G C C T C C T G G G C C C T T C C T Convolvulaceae Cuscuta campestris A T T C G T T A T G T - - C T C C C C T C T - - T G T G T G G T G C G - G G A G C A G A T T G T G G C C T C C T G G G C C C T T C C T Convolvulaceae Cuscuta umbrosa A T T C G T T A T G T - - C T C C C C T C T - - T G T G T G G T G C G - G G A G C A G A T T G T G G C C T C C T G G G C C C T T C C T Convolvulaceae Cuscuta umbrosa A T T C G T T A T G T - - C T C C C C T C T - - T G T G T G G T G C G - G G A G T A G A T T G T G G C C T C C T G G G C C C T T C C T Convolvulaceae Cuscuta gronovii A T T C G T T A T G T - - C T C C C C T C T - - T G T G T G G T G C G - G G A G T A G A T T G T G G C C T C C T G G G C C C T T C C T Convolvulaceae Cuscuta gronovii A A A T G T T T C G T - - C G T T C C T A A T C C T T T T T G T G A G - G G A C T G G T T C A T G G C C T C C C A G G C T A A G G T T Convolvulaceae Cuscuta indecora A A A T G T T T C G T - - C G T T C C T A A T C C T T T T T G T G A G - G G A C T G G T T C A T G G C C T C C C A G G C T A A G G T T Convolvulaceae Cuscuta indecora A C G T G A C G T G T C G C T C C C T T C T G G A T T T T T T T G G G A G G A G C G G A T A A T G T C C T C C C G T G C C T A T T G T Convolvulaceae Cuscuta approximata A C G T G A C G T G T C G C T C C C T T C T G G A T T T T T T T G G G A G G A G C G G A T A A T G T C C T C C C G T G C C T A T T G T Convolvulaceae Cuscuta approximata 70 75 80 85 90 95 100 Cuscuta indecora 69.9 Cuscuta indecora Cuscuta approximata Cuscuta approximata 68.2 Cuscuta umbrosa 99.7 Cuscuta umbrosa Cuscuta gronovii 91.1 Cuscuta gronovii Cuscuta campestris Cuscuta campestris DNA barcoding to visualise species links 78 80 82 84 86 88 90 92 94 96 98 76 100 Orobanchaceae Orobanche uniflora DAO 653751 85.2 Orobanchaceae Orobanche uniflora DAO 438357 Orobanchaceae Orobanche ludoviciana DAO 035116 Orobanchaceae Orobanche ludoviciana DAO 035107 Orobanchaceae Orobanche minor DAO 402190 83.6 99.7 Orobanchaceae Orobanche minor DAO 438307 Orobanchaceae Orobanche crenata DAO 322029 98.9 Orobanchaceae Orobanche crenata MICH 261 Orobanchaceae Orobanche hederae CAN 148927 96.6 99.9 Orobanchaceae Orobanche hederae UBC V26912 81.5 Orobanchaceae Orobanche cumana CAN 240529 99.7 Orobanchaceae Orobanche cumana MICH 3792 Orobanchaceae Orobanche cernua MICH 6119 Orobanchaceae Orobanche cernua MICH 7182 Orobanchaceae Orobanche ramosa DAO 284362 75.7 Orobanchaceae Orobanche ramosa DAO 438314 Orobanchaceae Striga forbesii US 633677 Orobanchaceae Striga forbesii US 3256690 95.2 Orobanchaceae Striga asiatica MICH 3722 Orobanchaceae Striga asiatica MICH 6432 Orobanchaceae BOT-15-015 93.3 99.7 Orobanchaceae Striga gesnerioides US 3006311 Orobanchaceae Striga gesnerioides US 2671711 99.2 Orobanchaceae Striga hermonthica US 97.5 Orobanchaceae Striga hermonthica US 1595603 Orobanchaceae Striga aspera US 37829 Orobanchaceae Striga aspera US 3107699 CFIA DNA barcode reference database in use TABLE 1. List of plant specimens submitted for identification Specimen submitted Morphological identification Barcode identification Specimen code Family Identified Species ITS2 trnH-psbA BOT-17-036 Poaceae Aegilops cylindrica Host Jointed goatgrass A.
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