Location: Mycology and Nematology Genetic Diversity and Biology LaboratoryTitle: PCR amplification of a long rDNA segment with one primer pair in agriculturally important nematodes
Submitted to: Journal of Nematology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/5/2019
Publication Date: 3/1/2019
Citation: Carta, L.K., Li, S. 2019. PCR amplification of a long rDNA segment with one primer pair in agriculturally important nematodes. Journal of Nematology. 51:e2019-26. https://doi.org/10.21307/jofnem-2019-026.
Interpretive Summary: Nematodes are microscopic worms that attack plants and cause billions of dollars of damage to crops and forest and ornamental trees. One problem in determining the extent of nematode damage is that the nematodes present in many areas may not be well known. Identification of nematodes often requires extensive microscopic observation and DNA profiling. Molecular tools called primers are used by scientists to generate DNA sequences that are used to identify nematodes. The longer the sequence information, the more accurate the identification can be. Some primers are better than others in producing the longest gene segment. Therefore, new primers were designed to amplify a fragment approximately three times the sequence length of previous primers. This was demonstrated in 17 different nematodes. The new primer tools and methods to use them are detailed so that nematodes important to agricultural trade can be more reliably identified from small amounts of DNA. This information will be used by agricultural researchers and diagnosticians in the U.S. and other countries.
Technical Abstract: Ribosomal DNA has been a reliable source of taxonomic and phylogenetic markers due to its high copy number in the genome and stable variation with few polymorphisms due to the homogenizing effect of concerted evolution. Typically, specific regions are amplified through polymerase chain reaction (PCR) and sequenced with very short nucleotide primer pairs that generate often incomplete and overlapping regions between adjacent segments of 18S, ITS1, 5.8S, ITS2, and 28S rDNA nucleotide sequences when combined in tandem. To improve the efficiency of this effort, a strategy for generating all these molecular sequences from a single fragment through PCR amplification of a large ribosomal 3.3-4.2 kb DNA target was developed using a new alternative 28S PCR primer (28S-CL-R) and other well-positioned and ribosomal-specific sequencing primers (including novel primers 18S-CL-F7, 18S-CL-R6, 18S-CL-R7, 18S-CL-F8, 5.8S-CL-F1, 5.8S-CL-R1, 28S-CL-F1, 28S-CL-R3, 28S-CL-F3, 28S-CL-R1, 28S-CL-F2). The D1 region between ITS2 and 28S boundaries and the flanking sequence between 18S and ITS1 boundaries were fully revealed in this large nucleotide segment. To demonstrate the value of this strategy the long rDNA segment was amplified and directly sequenced in 17 agriculturally important nematodes from the Tylenchida, Aphelenchida and Dorylaimida. The primers and their positions may be employed with traditional Sanger sequencing and with next-generation sequencing reagents and protocols.