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PCR amplification of a long rDNA segment with one primer pair in agriculturally important nematodes

L. K. Carta

L. K. Carta

Mycology and Nematology Genetic Diversity and Biology Laboratory, USDA, ARS, Henry A. Wallace Beltsville Agricultural Research CenterUSA
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Carta, L. K.
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S. Li

S. Li

Mycology and Nematology Genetic Diversity and Biology Laboratory, USDA, ARS, Henry A. Wallace Beltsville Agricultural Research CenterUSA
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Li, S.
  
03 cze 2019
PCR amplification of a long rDNA segment with one primer pair in agriculturally important nematodes's Cover Image
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Kategoria artykułu: Arts & Humanities
Data publikacji: 03 cze 2019
Zakres stron: 1 - 8
Otrzymano: 30 wrz 2018
DOI: https://doi.org/10.21307/jofnem-2019-026
Słowa kluczowe
© 2019 L. K. Carta et al., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.

Figure 1:

Position diagram of primers. (A): rDNA (AF248477), Meloidogyne artiellia; (B): rDNA (AY580056), Xiphinema americanum. (C): rDNA (X03680), Caenorhabditis elegans. Created with SnapGene software (GSL Biotech, Chicago, IL; available at snapgene.com) and assembled with Adobe Photoshop CC 2018 (Adobe Systems Incorporated, San Jose, CA).
Position diagram of primers. (A): rDNA (AF248477), Meloidogyne artiellia; (B): rDNA (AY580056), Xiphinema americanum. (C): rDNA (X03680), Caenorhabditis elegans. Created with SnapGene software (GSL Biotech, Chicago, IL; available at snapgene.com) and assembled with Adobe Photoshop CC 2018 (Adobe Systems Incorporated, San Jose, CA).

Figure 2:

PCR Amplification of 3.3 to 4.2 kb of rDNA from agriculturally important nematodes. M: DNA markers; 1: Heterodera orientalis 104F80; 2: Xiphinema sp. 104F83; 3: Hoplolaimus sp. 104G35; 4: Helicotylenchus sp. 104G36; 5: Meloidogyne incognita Me47; 6: Pratylenchus scribneri Pr1 and 7: Negative control.
PCR Amplification of 3.3 to 4.2 kb of rDNA from agriculturally important nematodes. M: DNA markers; 1: Heterodera orientalis 104F80; 2: Xiphinema sp. 104F83; 3: Hoplolaimus sp. 104G35; 4: Helicotylenchus sp. 104G36; 5: Meloidogyne incognita Me47; 6: Pratylenchus scribneri Pr1 and 7: Negative control.

Ribosomal primers used for PCR and sequencing_

Primers Direction Loci Sequence (5′-3′) PCR Sequencing References
18S-CL-F3 F 18S CTTGTCTCAAAGATTAAGCCATGCAT ✓ ✓ Carta and Li (2018)
D3B R 28S TCGGAAGGAACCAGCTACTA ✓ ✓ Nunn (1992)
28S-CL-R R 28S CAGCTACTAGATGGTTCGATTAGTC ✓ ✓ This study
18S-530F (530F) F 18S AAGTGTGGTGCCAGCAGCCGC ✓ Reverse complement of 530R
18S-530R (530R) R 18S GCGGCTGCTGGCACCACACTT ✓ Thomas et al. (2011)
1912R R 18S TTTACGGTCAGAACTAGGG ✓ Holterman et al. (2006)
18S-CL-R2 R 18S GTTGAGTCAAATTAAGCCGCA ✓ Carta and Li (2018)
18S-CL-F7 F 18S TGCGGCTTAATTTGACTCAAC ✓ This study
18S-CL-F2 F 18S CTGTGATGCCCTTAGATGTCC ✓ Carta and Li (2018)
18S-CL-R5 R 18S GCGGTGTGTACAAAGGGCAGGGAC ✓ Carta and Li (2018)
18S-CL-R6 R 18S ACCTTGTTACGACTTTTACTTCCTCTA ✓ ✓ This study
18S-CL-R7 R 18S ACCTTGTTACGACTTTTGCCCGGTTCA ✓ ✓ This study
18S-CL-F8 F 18S TGAACCGGGCAAAAGTCGTAACAAGGT ✓ This study
ITS-CL-F2 F ITS ATTACGTCCCTGCCCTTTGTA ✓ ✓ Carta and Li (2018)
5.8S-CL-F1 F 5.8S GATTCCATCATTCTAAGC ✓ This study
5.8S-CL-R1 R 5.8S ACCGCTTAGAATGATGGAATC ✓ This study
rDNA15.8S F 5.8S ACGAGCCGAGTGATCCACCG ✓ Cherry et al. (1997)
5.8SF F 5.8S CGGTGGATCACTCGGCTCGT ✓ Reverse complement of rDNA1.58S
AB28 R 28S ATATGCTTAAGTTCAGCGGGT ✓ Joyce et al. (1994)
28S-CL-F1 F 28S CTGAACTTAAGCATATCAGTAAGC ✓ This study
VRAIN 2R (V2R) R 28S TTTCACTCGCCGTTACTAAGGGAATC ✓ Vrain et al. (1992)
D2AR R 28S ACTTTCCCTCACGGTACTTGT ✓ Reverse complement of D2A
D2A F 28S ACAAGTACCGTGAGGGAAAGT ✓ Nunn (1992)
28S-CL-R3 R 28S GCAACTTTCCCTCACGGTACTTG This study
28S-CL-F3 F 28S AAGAGAGAGTTAAAGAGGACGTGAA ✓ This study
28S-CL-R1 R 28S ACTCCTTGGTCCGTGTTTCAAG ✓ This study
28S-CL-F2 F 28S CGACCCGTCTTGAAACAC ✓ This study
28S-1006rev (1006R) R 28S GTTCGATTAGTCTTTCGCCCCT ✓ Holterman et al. (2008)

Agriculturally important nematodes tested in this study_

Taxa Origin and locality PCR primer pair Ta (°C) Sequencing primers
Aphelenchoides fragariae Live specimen 104J12, provided by Dr. Paula Agudelo, Clemson University, Clemson, South Carolina 18S-CL-F3 and 28S-CL-R 50 530F, 530R, 1912R, 18S-CL-F2, 18S-CL-R2, 18S-CL-R5, ITS-CL-F2,28S-CL-F1, D2AR, 28S-CL-F3, 28S-CL-R1 and 28S-CL-R
Bursaphelenchus sp. Live specimen 104J7, isolated from walnut twig beetles, Washington 18S-CL-F3 and 28S-CL-R 57 530F, -530R, 18S-CL-R2, 18S-CL-F7,18S-CL-F2, 18S-CL-R5, 18S-CL-R7, 18S-CL-F8, ITS-CL-F2, rDNA15.8S, 28S-CL-F1, D2AR, 28S-CL-F3, 28S-CL-R1, 28S-CL-F2 and 1006R
Bursaphelenchus xylophilus Live specimen 104H46, isolated from Eastern white pine tree, New Hampshire 18S-CL-F3 and 28S-CL-R 57 530F, -530R, 18S-CL-R2, 18S-CL-F7,18S-CL-F2, 18S-CL-R5, 18S-CL-R7, 18S-CL-F8, ITS-CL-F2, rDNA15.8S, 28S-CL-F1, D2A, D2AR, 28S-CL-F3, 28S-CL-R1, 28S-CL-F2 and 1006R
Crossonema sp. Live specimen 104G38, isolated from the rhizosphere of bamboo, Beltsville, Maryland 18S-CL-F3 and D3B 57 530F, 530R, 1912R, 18S-CL-F2, 18S-CL-R2, 18S-CL-R5, ITS-CL-F2,28S-CL-F1, D2AR, 28S-CL-F3, 28S-CL-R1 and 28S-CL-R
Ditylenchus dipsaci Live specimen 85A5, obtained from French Iris in Wisconsin by USDA-APHIS-PPQ interception 18S-CL-F3 and 28S-CL-R 50 530R, 1912R,18S-CL-R2, 18S-CL-F7,18S-CL-F2, 18S-CL-R5, 18S-CL-R7, 18S-CL-F8, ITS-CL-F2, rDNA15.8S, AR28, V2R, 28S-CL-F1, 28S-CL-F3, D2AR, 28S-CL-R1, and 28S-CL-F2
Ditylenchus sp. Live specimen 85C1, isolated from the rhizosphere of alfalfa, Moab, Utah 18S-CL-F3 and 28S-CL-R 50 530R, 1912R,18S-CL-R2, 18S-CL-F7,18S-CL-F2, 18S-CL-R5, 18S-CL-R7, 18S-CL-F8, ITS-CL-F2, rDNA15.8S, 5.8SF, AB28, V2R, 28S-CL-F1, 28S-CL-F3, 28S-CL-R3, 28S-CL-R1, 28S-CL-F2, 1006R
Ecumenicus sp. Live specimen 85G11, isolated from the rhizosphere of alfalfa, St. George, Utah 18S-CL-F3 and D3B 50 530R, 530F,18S-CL-R2, 18S-CL-F7,18S-CL-F2, 18S-CL-R5, 18S-CL-R6, ITS-CL-F2, rDNA15.8S, 5.8SF, V2R, 28S-CL-F1, D2AR, D2A, 28S-CL-R3, 28S-CL-R1, 28S-CL-F2, 1006R
Helicotylenchus sp. Live specimen 104G36, isolated from the rhizosphere of bamboo, Beltsville, Maryland 18S-CL-F3 and D3B 57 530R, 530F,1912R, 18S-CL-R2, 18S-CL-F7,18S-CL-F2, 18S-CL-R5, 18S-CL-F8, ITS-CL-F2, 5.8SF, V2R, 28S-CL-F1, D2AR, 28S-CL-R1, 28S-CL-F2, 28S-CL-R
Heterodera glycines Live J2 specimen Hg20, from the isolate, NL1-RHp originally was collected in on the east shore of Maryland and raised on soybean (G. max, cv. Kent) in sand-filled beakers 18S-CL-F3 and D3B 57 530R, 1912R, 18S-CL-R2, 18S-CL-F7,18S-CL-F2, 18S-CL-R7, 18S-Cl-F8, rDNA15.8S, 5.8SF, AB28, 28S-CL-F1, D2A, 28S-CL-F3, 28S-CL-R1, 28S-CL-F2, 1006R, and 28S-CL-R
Heterodera orientalis Live J2 specimen 104F80, isolated from the cyst in the rhizosphere of Miscanthus, Beltsville, Maryland. 18S-CL-F3 and D3B 57 530R, 530F, 1912R, 18S-CL-R2, ,18S-CL-F2, 18S-CL-R5, ITS-CL-F2, 28S-CL-F1, D2AR, 28S-CL-F3, 28S-CL-R1, 28S-CL-F2, 28S-CL-R
Hoplolaimus sp. Live specimen 104G35, isolated from the rhizosphere of bamboo, Beltsville, Maryland 18S-CL-F3 and D3B 57 530R, 530F, 1912R, 18S-CL-R2, 18S-CL-F2, 18S-CL-R5, 18S-CL-F8, ITS-CL-F2, rDNA15.8S, 5.8SF, AB28, V2R, 28S-CL-F1, D2A, 28S-CL-F3, 28S-CL-R3, 28S-CL-R1, and 28S-CL-F2
Litylenchus sp. Live specimen 104H88, isolated from the leaf of beech, Perry, Ohio 18S-CL-F3 and 28S-CL-R 50 530R, 530F, 1912R, 18S-CL-R2, 18S-CL-F2, 18S-CL-R5, 18S-CL-R7, ITS-CL-F2, rDNA15.8S, V2R, 28S-CL-F3, 28S-CL-R1, 28S-CL-F2, 1006R and 28S-CL-R
Meloidogyne incognita Live J2 specimen Me47 from the isolate, RKN Race 1, originally was collected in Maryland and maintained with ‘PA-136’ pepper in greenhouse pots 18S-CL-F3 and D3B 57 530R, 530F, 1912R, 18S-CL-R2, 18S-CL-F2, 18S-CL-F7, 18S-CL-R5, 18S-CL-R7, ITS-CL-F2, 5.8SF, V2R, 28S-CL-R1, 28S-CL-F2 and 1006R
Pratylenchus scribneri Live specimen Pr1 from a culture maintained with corn root explant; originally collected from soil in Beltsville, Maryland 18S-CL-F3 and D3B 57 18S-CL-F3, 530R, 530F, 1912R, 18S-CL-R2, 18S-CL-F2, 18S-CL-F7, 18S-CL-R5, 18S-CL-R7, ITS-CL-F2, rDNA15.8S, 5.8SF, Ab28, V2R, D2Ar, D2A, 28S-CL-F3, 28S-CL-R3, 28S-CL-R1, 28S-CL-F2 and 28S-CL-R
Radopholus similis Live specimen 31G1, obtained by USDA – APHIS – PPQ interception from Anthurium in Kurtistown, Hawaii 18S-CL-F3 and 28S-CL-R 50 530R, 530F,1912R, 18S-CL-R2, 18S-CL-F2, 18S-CL-F7, 18S-CL-R5, 18S-CL-R7, ITS-CL-F2, rDNA15.8S, 5.8SF, AB28, V2R, D2AR, 28S-CL-F1, 28S-CL-F3, 28S-CL-R1, 28S-CL-F2 and 1006R
Xiphinema sp. Live specimen 06D2, isolated from soil in Clarksville, Maryland 18S-CL-F3 and D3B 50 18S-CL-F3,530R, 530F,1912R, 18S-CL-R2, 18S-CL-F2, 18S-CL-F7, 18S-CL-R5, 18S-CL-R6, ITS-CL-F2, 5.8S-CL-F1, 5.8S-CL-R1(XitsS3), V2R, D2AR,D2A, 28S-CL-F3, 28S-CL-R1, 28S-CL-F2, and 1006R
Xiphinema sp. Live specimen 104F83, isolated from the rhizosphere of bamboo, Beltsville, Maryland 18S-CL-F3 and D3B 57 18S-CL-F3, 530R, 530F,1912R, 18S-CL-R2, 18S-CL-F2, 18S-CL-F7, 18S-CL-R5, 18S-CL-R6, ITS-CL-F2, 5.8SF, 5.8S-CL-R1(Xist3), V2R, D2AR, 28S-CL-F2, 1006R and 28S-CL-R
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Angielski
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Dziedziny czasopisma:
Nauki biologiczne, Nauki biologiczne, inne
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