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

Carta, L. K.; Li, S.

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

Journal of Nematology

Volume 51 (2019): Issue 1 (January 2019)

By:

L. K. Carta and S. Li

Open Access

|Jun 2019

Figures & Tables

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).

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