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

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) with multiple 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 at once through PCR amplification of a large ribosomal 3.3 to 4.2 kb DNA target was developed using primer 18S-CL-F3 paired with D3B or 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, and 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.

Eukaryotic nuclear ribosomal DNA (rDNA) is arranged in tandem repeat arrays in the genome. Each repeat unit consists of one copy of small subunit (SSU) 18S, internal transcribed spacers (ITS1 and ITS2), 5.8S, and large subunit (LSU) 28S rDNA, and is separated by an external transcribed spacer (EST) and an intergenic spacer (IGS) (Hillis and Dixon, 1991). The copy number of the repeats within most eukaryotic genomes is high, which provide large quantities of template DNA for PCR. In Caenorhabditis spp., for instance, the rDNA copy number was estimated to be as many as 56 to 323 copies within their genomes (Bik et al., 2013). Second, the rDNA polymorphisms among the repeat units are very low within the genome due to concerted evolution (Liao, 1999).
These two features make rDNA particularly well suited for taxonomic identification, phylogenetic analysis, and barcoding for nematodes (Blaxter et al., 1998;Floyd et al., 2002;Holterman et al., 2006;Megen et al., 2009;Rodrigues Da Silva et al., 2010). As a result, there have been more than 300,000 nematode rDNA sequences published in GenBank to date. One of the most important means of determining these rDNA sequences is the amplification of the target rDNA loci by polymerase chain reaction (PCR). Usually 18S, 28S, and ITS are amplified separately with different PCR primer pairs; one or more PCR primer pairs are used to amplify 18S to near full length (Carta and Li, 2018), one pair for ITS1, 5.8S, and ITS2 (Ferris et al., 1993;Vrain, 1993;Joyce et al., 1994), and one pair for the D2D3 segment of 28S (Nunn, 1992). This multiple-pair approach is time-consuming and cost-ineffective for the amplification of this long target of approximately, 3.3 to 4.2 kb in length from 18S, ITS1, 5.8S, ITS2 to the D3 of 28S; furthermore, from a probabilistic point of view, the more primer pairs that are applied to amplify this long target, the lower the success rate of the amplification will be. Therefore, minimizing the number of primer pairs is a key to successfully amplifying this long target. In this short technical note, we have tested the PCR amplification of this 3.3 to 4.2 kb rDNA target with one ribosomal primer pair in 17 agriculturally important nematodes and sequenced the resulting amplicons directly with well-positioned and ribosomal-specific sequencing primers.

DNA extraction
Live J2 from Heterodera spp. and Meloidogyne incognita and live adult nematodes from other taxa described in Table 1 were selected for this study. Template DNA was prepared in the format of one nematode per tube by using freeze-thaw lysis. Before the extraction, the nematodes were washed twice with molecular biology grade water to remove any micro contaminants attached to their bodies. A clean single nematode was picked and transferred to a 0.2 ml PCR tube containing 24 µl of extraction buffer (10 mM Tris pH 8.2, 2.5 mM MgCl 2 , 50 mM KCl, 0.45% TWEEN 20, and 0.05% gelatin, Williams et al., 1992). The tube was submerged in liquid nitrogen for 10 to 15 sec and then placed at 95°C for 2 min in a C1000 Touch TM thermal cycler (Bio-Rad Laboratories, Hercules, CA). This rapid physical disruption was repeated once. Then the tube was subjected to the third cycle of freezing in liquid nitrogen for 10 to 15 sec and then was slow-thawed at room temperature. The thawed sample was lysed with 1 µl of proteinase K (800 U/ml, Sigma-Aldrich, St. Louis, MO) at 60°C for 60 min, followed by 95°C for 15 min to deactivate the proteinase K. At least three single nematodes from each taxon were picked for the individual DNA extraction. All resulting lysates were stored in a −20°C freezer until needed. The DNA extracts from Ditylenchus, Ecumenicus, and Radopholus were prepared previously by using mechanical lysis (Carta et al., 2010).

Results
The forward PCR primer for this long rDNA target was 18S-CL-F3, the reverse primer was D3B and a newly designed 28S primer, 28S-CL-R, was applied as well (Table 2). Figure 2 shows that this long rDNA target was amplified by PCR in six taxa from agriculturally important nematodes. The internal sequencing primers with reading overlaps (Fig. 1) were tested by using cycling amplification. The rDNA sequences resulted from Helicotylenchus sp. 104G36 (MK292128), Heterodera orientalis 104F80 (MK292130), Hoplolaimus sp. 104G35 (MK292131), Meloidogyne incognita Me47 (MK292132), Pratylenchus scribneri Pr1(MK292133), and Xiphinema sp. 104F83 (MK292136) with high coverage in each base were deposited to GenBank. Additionally, another 11 agriculturally important nematodes were also tested by using the same approach described above when they became available, and the resulting sequences with Accession No   and MK292135 for Xiphinema sp. 06D2 were deposited to GenBank as well.

Discussion
An approximate base-pair match between primer and template DNA is required for Taq DNA polymerase to begin an efficient PCR amplification cycle (Watson, 1971;Kwok et al., 1994;Wu et al., 2009;Wright et al., 2014). The taxonomic universality of 18S-CL-F3, the forward primer selected for the single primer pair approach, has been presented previously from taxa in Tylenchida and Dorylaimida (Carta and Li, 2018). We have not seen any failures of PCR amplification from Note: F: Forward; R: Reverse; √: Applicable.  the taxa tested with 18S-CL-F3 since it was designed in our lab. The D3B segment primer was selected because it is a universal reverse primer for amplifying the D2D3 segment of 28S across Nematoda (Nunn, 1992). Primer 28S-CL-R was also designed as a substitute for D3B. The primer pair 18S-CL-F3 and either D3B or 28S-CL-R is the primary key to the success of the single primer pair approach achieved in this study. To our knowledge, this is the first report of using a single primer pair to amplify this long rDNA target in diverse agriculturally important nematodes. Although this long rDNA target was amplified successfully with a traditional Taq DNA polymerase used in the single primer pair approach, it should be noted that Taq DNA polymerase lacks 3′-5′ exonuclease activity and is incapable of proofreading any misincorporated nucleotides during PCR (Tindall and Kunkel, 1988). This inability could make the Taq dissociate from its template DNA before the extension is completed and subsequently limits the size of the amplicon (Arezi et al., 2003). Therefore, it is desirable that thermal proofreading DNA polymerase, Pfu, Vent or others, along with a Taq or a blend of both enzymes be employed in the single primer pair approach when the PCR amplification of the long target becomes difficult (Barnes, 1994;Cheng et al., 1995). Strong non-specific amplicon bands in the specimens 104G35 and Me47 (Lane 3 and Lane 5 in Figure 2, respectively) were observed, however, their sequence reads were not interrupted by the non-target amplicons (data not shown). This is because the ribosomal internal sequencing primers applied in this study can only recognize the rDNA amplicon that has these primer binding sites. Therefore, choosing the internal sequencing primers is critical for the direct DNA sequencing in the single primer pair approach. Primer 530R (Table 2) was se-lected and 28S-CL-F2 designed specifically to read the 5′-end and 3′-end of this 3.3 to 4.2 kb rDNA amplicon, respectively, during cycling with BigDye® reagents. These two primers were particularly useful for sequencing because the PCR primers may not be used as sequencing primers when non-specific bands (amplicons) occur. Both 18S-CL-R7 and 18S-CL-R6 were designed as sequencing primers initially for Tylenchida and Dorylaimida, respectively. Moreover, they could also be paired with 18S-CL-F3 to amplify the 18S in near-full length as needed; and 28S-CL-R could be paired with ITS-CL-F2 for either the amplification of ITS, or for 28S (D1D2D3) or both to meet different goals.
In this study, we demonstrated the PCR amplification of this 3.3 to 4.2 kb rDNA in 17 agriculturally important nematodes using the single primer pair approach. The taxonomic coverage by these two single primer pairs revealed in this study suggests that they may also be valid for other plant-parasitic nematode taxa in Tylenchida and Dorylaimida. Additionally, this ability was seen in several taxa in Rhabditida with the 18S-CL-F3 and 28S-CL-R pair (data not shown). This study also provides the internal ribosomal sequencing primers that are well positioned in the target rDNA (Table 2 and Fig. 1) with high base coverages to acquire the high quality rDNA sequences spanning from 18S in near full length, ITS1 in full length, 5.8S in full length, ITS2 in full length, to the D1, D2, and D3 segments of 28S at once, which would facilitate deep phylogenetic analysis and accurate taxonomic identification by using individual specimens. Particularly, the D1 segment between ITS2 and 28S and the flanking sequence between 18S and ITS1 were fully revealed by the single primer pair approach, while they are a blind spot in the multiple primer pair approach. These ribosomal primer pairs could also be utilized for meta-barcoding by targeting this long rDNA target from environmental nematode DNA samples. The resulting amplicons could be sequenced using different Next Generation Sequencing platforms such as PacBio (Pacific Bio-Sciences, Menlo Park, CA) and Nanopore (Oxford Nanopore Technologies, Oxford, UK) for long reads.