The term DNA-barcoding has multiple definitions. The earliest mention of barcoding in nematology was in 1998 by Dr Mark Blaxter, then of Edinburgh University, referring to the “(d)evelopment of a molecular barcode system for soil nematode identification” in the first volume of the Natural Environment Research Council Soil Biodiversity Newsletter (
Nematology was slow to adopt this formalized version of barcoding, perhaps due to poor amplification with the original “Folmer” primer sets (Folmer et al., 1994). Now multiple primer sets for amplification of nematode cytochrome oxidase c subunit 1 (COI) are available (Derycke et al., 2005, 2010; Prosser et al., 2013; Kiewnick et al., 2014; Powers et al., 2014; Janssen et al., 2016). These primer sets typically have limited taxonomic scope with amplifications specific for genera or in some cases extending across families and superfamilies (Powers et al., 2014). The objective of this study is to present a primer set used for the amplification of 721 to 724 bp of COI sequence from
Most of the specimens DNA barcoded in this study were either specimens submitted to the UNL Nematology Diagnostics Clinic, specimens contributed by colleagues, or specimens collected during grant funded surveys (NSF projects DEB-1145440; USDA Multistate Project W3186).
The primer set for amplification of the COI gene region were:
COI-F5-Mel – 5′-TGATTGATTTAGGTTCTGGAACTKSWTGAAC-3′
COI-R9-Mel – 5′-CATAATGAAAATGGGCAACAACATAATAAGTATC-3′
After removal of the primer sequences, amplification products from the
Nematodes amplified at the UNL Nematology Laboratory were individually smashed in 18 ul of sterile H20 with a transparent microfuge micropipette tip on a coverslip and added to a 0.5 ml microfuge tube. Nematode lysate was either amplified immediately or stored at −20°C. Amplification conditions were as follows: denaturation at 94°C for 5 min, followed by 45 cycles of denaturation at 94°C for 30 sec, annealing at 48.0°C for 30 sec, and extension at 72°C for 90 sec with a 0.5° per second ramp rate to 72°C. A final extension was performed at 72 °C for 5 min as described by Powers et al. (2014) and Olson et al. (2017). Polymerase chain reaction (PCR) products were separated and visualized on 1% agarose using 0.5XTBE and stained with ethidium bromide. PCR products of sufficiently high quality were cleaned and sent for sequencing of both strands by University of California–Davis DNA sequencing facility.
Nucleotide sequences have been submitted to GenBank (accession numbers MH128384–MH128585) and the Barcode of Life Database (BOLD).
Phylogenetic trees were constructed under ML and Neighbor Joining (NJ) criteria in MEGA version 6. Sequences were edited using CodonCode Aligner version 7.1 (
Figure 1 displays a ML tree of 322
COI sequence collection data for groups 2 to 19.
NID | Group | Species | Locality | Host/habitat | GenBank accession # | |
---|---|---|---|---|---|---|
P203060 | 3 |
|
Florida | Ornamentals-Nursery | MH128522 | |
P179069 | 3 |
|
Florida | Ornamentals-Nursery | MH128519 | |
P179070 | 3 |
|
Florida | Ornamentals-Nursery | MH128520 | |
P196090 | 3 |
|
Florida | Ornamentals-Nursery | MH128521 | |
P210013 | 3 |
|
Florida | Ornamentals-Nursery | MH128523 | |
P210014 | 3 |
|
Florida | Ornamentals-Nursery | MH128524 | |
P210071 | 3 |
|
Florida | Ornamentals-ursery | MH128529 | |
P210059 | 3 |
|
Florida | Ornamentals-Nursery | MH128527 | |
P210057 | 3 |
|
Florida | Ornamentals-Nursery | MH128525 | |
P210058 | 3 |
|
Florida | Ornamentals-Nursery | MH128526 | |
P210072 | 3 |
|
Florida | Ornamentals-Nursery | MH128530 | |
P210065 | 3 |
|
Florida | Ornamentals-Nursery | MH128528 | |
N4314 | 4 |
|
Lance Rosier Unit, BITHa | Loblolly pine | MH128531 | |
N4321 | 4 |
|
Lance Rosier Unit, BITHa | Loblolly pine | MH128532 | |
N4379 | 5 |
|
Cove Mtn. Trail, GRSMb | Chestnut | MH128537 | |
N4388 | 5 |
|
Cove Mtn. Trail, GRSMb | Chestnut | MH128538 | |
N2110 | 5 |
|
GWMPc | Fort Marcy earthworks | MH128533 | |
N3952 | 5 |
|
Turkey Creek, BITHa | Baygall community | MH128534 | |
N4285 | 5 |
|
Canyonlands South, BITHa | Magnolia | MH128535 | |
N4291 | 5 |
|
Canyonlands South, BITHa | Magnolia | MH128536 | |
P214008 | 6 |
|
Dona Ana County, New Mexico | Pecan | MH128540 | |
P214010 | 6 |
|
Dona Ana County, New Mexico | Pecan | MH128542 | |
P214009 | 6 |
|
Dona Ana County, New Mexico | Pecan | MH128541 | |
N2338 | 6 |
|
Neches Bottoms Unit, BITHa | Sandbar | MH128539 | |
P121054 | 7 |
|
Gasconade County, Missouri | Peony | MH128568 | |
P200031 | 7 |
|
Cass County, Nebraska | Nursery | MH128577 | |
N163 | 7 |
|
Nebraska | Wheat | MH128543 | |
P200018 | 7 |
|
Daggett County, Utah | Grass pasture | MH128575 | |
P200019 | 7 |
|
Daggett County, Utah | Grass pasture | MH128576 | |
P178064 | 7 |
|
Oregon | Potato | MH128570 | |
P200032 | 7 |
|
Colfax County, Nebraska | Nursery | MH128578 | |
N1376 | 7 |
|
Fremont County, Wyoming | Red bean | MH128558 | |
N1377 | 7 |
|
Fremont County, Wyoming | Red bean | MH128559 | |
P179068 | 7 |
|
New York | GH | MH128572 | |
P178063 | 7 |
|
Oregon | Potato | MH128569 | |
P222083 | 7 |
|
Portales, New Mexico | GH culture | MH128579 | |
P222084 | 7 |
|
Portales, New Mexico | GH culture | MH128580 | |
N1448 | 7 |
|
GWMPc | Waterfowl sanctuary | MH128560 | |
N857 | 7 |
|
Goshen County, Wyoming | Potato | MH128554 | |
N859 | 7 |
|
Goshen County, Wyoming | Potato | MH128555 | |
N860 | 7 |
|
Goshen County, Wyoming | Potato | MH128556 | |
N861 | 7 |
|
Goshen County, Wyoming | Potato | MH128557 | |
P200001 | 7 |
|
Hot Springs County, Wyoming | Alfalfa | MH128573 | |
P200002 | 7 |
|
Hot Springs County, Wyoming | Alfalfa | MH128574 | |
N4124 | 7 |
|
Wyoming | – | MH128561 | |
N318 | 7 |
|
Idaho | Potato | MH128544 | |
N497 | 7 |
|
California | – | MH128551 | |
N489 | 7 |
|
California | – | MH128550 | |
N498 | 7 |
|
California | – | MH128552 | |
P179054 | 7 |
|
Rhode Island | GH culture | MH128571 | |
N320 | 7 |
|
Idaho | Potato | MH128545 | |
N358 | 7 |
|
Idaho | Potato | MH128546 | |
N359 | 7 |
|
Idaho | Potato | MH128547 | |
N421 | 7 |
|
Carbon County, Montana | Alfalfa | MH128548 | |
N422 | 7 |
|
Carbon County, Montana | Alfalfa | MH128549 | |
N856 | 7 |
|
Goshen County, Wyoming | Potato | MH128553 | |
N7097 | 7 |
|
Nebraska | Alfalfa | MH128562 | |
N7098 | 7 |
|
Nebraska | Alfalfa | MH128563 | |
N7100 | 7 |
|
Nebraska | Alfalfa | MH128565 | |
N7099 | 7 |
|
Nebraska | Alfalfa | MH128564 | |
N8595 | 7 |
|
Chalti, Nepal | Pine forest | MH128566 | |
N8612 | 7 |
|
Chalti, Nepal | Pine forest | MH128567 | |
N4222 | 9 |
|
Canyonlands South, BITHa | Beech | MH128581 | |
N4229 | 9 |
|
Canyonlands South, BITHa | Beech | MH128582 | |
N8431 | 9 |
|
Canyonlands South, BITHa | Beech | MH128584 | |
N8433 | 9 |
|
Canyonlands South, BITHa | Beech | MH128585 | |
N8283 | 9 |
|
Mt. St. Hilaire, Quebec, Canada | Hardwood forest | MH128583 | |
N4431 | 10 |
|
Cove Mtn. Trail, GRSMb | Chestnut | MH128463 | |
N4496 | 10 |
|
Cove Mtn. Trail, GRSMb | Chestnut | MH128464 | |
N4497 | 11 |
|
Cove Mtn. Trail, GRSMb | Chestnut | MH128465 | |
N8084 | 12 |
|
Purchase Knob, GRSMb | Chestnut | MH128468 | |
N8121 | 12 |
|
Cataloochee, GRSMb | Oak | MH128470 | |
N8058 | 12 |
|
Cataloochee, GRSMb | Chestnut | MH128467 | |
N8012 | 12 |
|
Cataloochee, GRSMb | Chestnut | MH128466 | |
N8111 | 12 |
|
Cataloochee, GRSMb | Oak | MH128469 | |
N1479 | 13 |
|
Roy E. Larsen Sandylands, BITHa | Baygall community | MH128471 | |
N3969 | 13 |
|
Turkey Creek, BITHa | Baygall community | MH128472 | |
P129052 | 14 |
|
Costa Rica | Rice | MH128473 | |
P129054 | 14 |
|
Costa Rica | Rice | MH128474 | |
P169011 | 14 |
|
Florida | Purple nutsedge | MH128475 | |
N214 | 15 |
|
Nicaragua | Coffee | MH128477 | |
N215 | 15 |
|
Nicaragua | Coffee | MH128478 | |
N213 | 15 |
|
Nicaragua | Coffee | MH128476 | |
P199069 | 16 |
|
Sanpete County, Utah | Grass | MH128480 | |
P199071 | 16 |
|
Sanpete County, Utah | Grass | MH128481 | |
P199072 | 16 |
|
Sanpete County, Utah | Grass | MH128482 | |
N326 | 16 |
|
Idaho | Potato | MH128479 | |
P192084 | 17 |
|
Scotland | Genomic DNA | MH128507 | |
P119032 | 17 |
|
New Mexico | Culture | MH128488 | |
P115026 | 17 |
|
Fort Garland, Colorado | Soil sample | MH128487 | |
P122010 | 17 |
|
Colorado | Soil sample | MH128489 | |
P122047 | 17 |
|
Colorado | Soil sample | MH128490 | |
P124056 | 17 |
|
Commercial | Potato | MH128491 | |
P124057 | 17 |
|
Commercial | Potato | MH128492 | |
P124059 | 17 |
|
Commercial | Potato | MH128493 | |
N7145 | 17 |
|
Elko County, Nevada | Potato | MH128483 | |
N7147 | 17 |
|
Elko County, Nevada | Potato | MH128484 | |
N7148 | 17 |
|
Elko County, Nevada | Potato | MH128485 | |
N7149 | 17 |
|
Elko County, Nevada | Potato | MH128486 | |
P173100 | 17 |
|
Commercial | Potato | MH128494 | |
P174001 | 17 |
|
Commercial | Potato | MH128495 | |
P175068 | 17 |
|
Idaho | Potato | MH128496 | |
P175069 | 17 |
|
Idaho | Potato | MH128497 | |
P175070 | 17 |
|
Idaho | Potato | MH128498 | |
P175071 | 17 |
|
Idaho | Potato | MH128499 | |
P177092 | 17 |
|
Texas | Potato | MH128500 | |
P177094 | 17 |
|
Texas | Potato | MH128501 | |
P177098 | 17 |
|
Texas | Potato | MH128502 | |
P192011 | 17 |
|
Commercial | Potato | MH128504 | |
P192012 | 17 |
|
Commercial | Potato | MH128505 | |
P192013 | 17 |
|
Commercial | Potato | MH128506 | |
P211088 | 17 |
|
Oregon | Potato | MH128508 | |
P211089 | 17 |
|
Oregon | Potato | MH128509 | |
P212013 | 17 |
|
California | Potato | MH128510 | |
P212014 | 17 |
|
California | Potato | MH128511 | |
P212015 | 17 |
|
California | Potato | MH128512 | |
P212016 | 17 |
|
California | Potato | MH128513 | |
P213039 | 17 |
|
Washington | Potato | MH128514 | |
P213040 | 17 |
|
Washington | Potato | MH128515 | |
P221087 | 17 |
|
New Mexico | Potato | MH128518 | |
P215032 | 17 |
|
Washington | Potato | MH128517 | |
P178028 | 17 |
|
Commercial | Potato | MH128503 | |
P215031 | 17 |
|
Washington | Potato | MH128516 |
aBITH=Big Thicket National Preserve, Texas.
bGRSM=Great Smoky Mountains National Park, Tennessee and North Carolina.
cGWMP=George Washington Memorial Parkway, Virginia.
COI sequences included in group 1.
NID | Species | Locality | Host/Habitat | GenBank accession # |
---|---|---|---|---|
N137 |
|
Hawaii | Pineapples | MH128384 |
N138 |
|
Hawaii | Pineapples | MH128385 |
N7067 |
|
Charleston, Missouri | Soybean | MH128414 |
N7066 |
|
Charleston, Missouri | Soybean | MH128413 |
N7065 |
|
Charleston, Missouri | Soybean | MH128412 |
N5777 |
|
Nebraska | Conservatory | MH128410 |
N5775 |
|
Nebraska | Conservatory | MH128409 |
N5771 |
|
Nebraska |
|
MH128408 |
N3836 |
|
Nebraska | Banana | MH128407 |
N2668 |
|
Sonora, Mexico | Grapevine | MH128406 |
N2667 |
|
Sonora, Mexico | Grapevine | MH128405 |
N2666 |
|
Sonora, Mexico | Grapevine | MH128404 |
N2665 |
|
Sonora, Mexico | Grapevine | MH128403 |
N2664 |
|
Sonora, Mexico | Grapevine | MH128402 |
N2663 |
|
Sonora, Mexico | Grapevine | MH128401 |
N2662 |
|
Sonora, Mexico | Grapevine | MH128400 |
N2661 |
|
Sonora, Mexico | Grapevine | MH128399 |
N2659 |
|
Florida | Peanuts | MH128397 |
N7068 |
|
Clarkton, Missouri | Soybean | MH128415 |
N7069 |
|
Clarkton, Missouri | Soybean | MH128416 |
N7070 |
|
Clarkton, Missouri | Soybean | MH128417 |
N7072 |
|
Clarkton, Missouri | Soybean | MH128418 |
N7073 |
|
Clarkton, Missouri | Soybean | MH128419 |
N7075 |
|
Clarkton, Missouri | Soybean | MH128420 |
N8309 |
|
Charleston, Missouri | Soybean | MH128421 |
P118094 |
|
Missouri | Potato | MH128424 |
P120058 |
|
Arizona | Culture | MH128425 |
P120059 |
|
Arizona | Culture | MH128426 |
P121032 |
|
Mississippi | Potato | MH128427 |
P121058 |
|
Gasconade County, Missouri | Daylily | MH128428 |
P121060 |
|
Moniteau County, Missouri | Daylily | MH128429 |
P156046 |
|
Florida | GH culture | MH128430 |
P156048 |
|
Florida | GH culture | MH128431 |
P158036 |
|
Alachua County, Florida | – | MH128432 |
P158037 |
|
Alachua County, Florida | – | MH128433 |
P160024 |
|
Alachua County, Florida | – | MH128434 |
P160025 |
|
Alachua County, Florida | – | MH128435 |
P160075 |
|
Alachua County, Florida | – | MH128437 |
P167014 |
|
– | – | MH128438 |
P167019 |
|
– | – | MH128439 |
P167020 |
|
– | – | MH128440 |
P167021 |
|
– | – | MH128441 |
P176014 |
|
Missouri | Culture | MH128443 |
P178075 |
|
Texas | Potato | MH128444 |
P195088 |
|
– | – | MH128445 |
P195089 |
|
– | – | MH128446 |
P196023 |
|
– | – | MH128447 |
P196024 |
|
– | – | MH128448 |
P196025 |
|
– | – | MH128449 |
P202009 |
|
Israel | Culture | MH128450 |
P229051 |
|
Florida | Culture | MH128451 |
P229053 |
|
Florida | Culture | MH128452 |
P229056 |
|
Florida | Culture | MH128453 |
P233011 |
|
– | Coffee | MH128457 |
P233014 |
|
– | Coffee | MH128458 |
P234004 |
|
Morocco | – | MH128459 |
P234005 |
|
Morocco | – | MH128460 |
P234006 |
|
Morocco | – | MH128461 |
P234007 |
|
Morocco | – | MH128462 |
P73085 |
|
Bonita, Arizona | Pinto beans | MH128422 |
P73088 |
|
Bonita, Arizona | Pinto beans | MH128423 |
N5796 |
|
Ash Meadows NWR, Nevada | – | MH128411 |
P230069 |
|
West Virginia | Culture | MH128454 |
P230095 |
|
West Virginia | Culture | MH128456 |
P230070 |
|
West Virginia | Culture | MH128455 |
N2660 |
|
Florida | Peanut | MH128398 |
P167027 |
|
– | – | MH128442 |
N329 |
|
North Dakota | Potato | MH128386 |
N330 |
|
North Dakota | Potato | MH128387 |
N331 |
|
North Dakota | Potato | MH128388 |
N332 |
|
North Dakota | Potato | MH128389 |
N333 |
|
North Dakota | Potato | MH128390 |
N334 |
|
North Dakota | Potato | MH128391 |
N335 |
|
North Dakota | Potato | MH128392 |
N336 |
|
North Dakota | Potato | MH128393 |
N337 |
|
North Dakota | Potato | MH128394 |
N348 |
|
North Dakota | Potato | MH128395 |
N351 |
|
North Dakota | Potato | MH128396 |
P160071 |
|
Alachua, Florida | Culture | MH128436 |
Groups 1 to 3 form a clade characterized by the loss of a single amino acid (3 bp) resulting in a 721 bp sequenced region. This shared deletion unites
Outside of clades 1-3 there are 11 other described species represented by a minimum of a single COI sequence.
There are seven groups labeled as unnamed, all with sequence derived from j2 stage specimens except for N4431 and N4496 which were males collected from native chestnut (
Measurements of j2 Meloidogyne specimens from unnamed COI haplotype groups and reference species.
Haplotype group/species |
|
Length | Tail length | Stylet length |
|
|
|
---|---|---|---|---|---|---|---|
Unnamed 4 | 2 | 441 (430–452) | 42 | 17 | 24.3 (22.7–25.9) | 3.8 (3.7–3.9) | 10.5 (10.3–10.8) |
Unnamed 5 | 5 | 431 (406–460) | 47 (40–53) | 14 (13–15 | 25.4 914.4–30.8) | 3.9 (3.0–4.8) | 9.3 (8.0–10.1) |
Unnamed 9 | 5 | 393 (380–405) | 40 (38–44) | 15 (15–16) | 26.0 (25.5–26.7) | 4.0 (3.3–4.4) | 9.7 (9.2–10.1) |
Unnamed 11 (Singleton A) | 1 | 384 | 42 | 15 | 27.5 | 3.5 | 9.1 |
Unnamed 12 | 5 | 353 (339–379) | 41 (38–43) | 15 (14–15) | 22.7 (21.2–25.0) | 3.5 (3.3–4.0) | 8.6 (8.0–8.9) |
Unnamed 13 | 2 | 490 (439–541) | 59 (57–62) | 14 | 30.7 (30.4–31) | 3.9 (3.8–4.0) | 8.2 (7.7–8.6) |
|
10 | 370 (350–430) | – | – | 22 (21–24) | – | 8 (8–9) |
|
30 | 434 (376–493) | 44 (37–53) | 12.8 (11.4–14.1) | 25.7 (21.8–29.1) | – | 9.8 (7.5–11.8) |
|
70 | 501 (443–576) | 47 (40–56) | 11.6 (11.2–12) | 26 (21–30) | 3.1 | 10.7 (9.5–12 |
|
70 | 467 (411–541) | 46 (39–52) | 11.1 (10.2–11.6) | 30 (23–39) | 2.6 | 10 (7–13) |
|
23 | 416 (392–457) | 39.7 (31.6–45.1) | 14.6 (13.8–16.6) | 26 (22–29) | 7.1 (6.7–7.8) | 10.5 (9.5–13.5) |
Maximum likelihood tree of 322
Selected
The COI gene region used as a diagnostic marker in this study appears to discriminate many of the described species of