Improved 18S small subunit rDNA primers for problematic nematode amplification

Abstract

The 18S small subunit (SSU) ribosomal DNA sequence is one of the most useful molecular loci for identification and phylogeny reconstruction of agriculturally important nematodes. Various pairs of universal primers have been developed in the past to amplify short and long nematode sequences. However, certain nematode taxa were not readily amplified and/or sequenced with the existing primer tools. Frequently, the center region of a roughly 1,000 nucleotide segment would be lost. Therefore new primers were developed based on a very large 276 taxon alignment of 124 agriculturally important nematode species, and tested on problematic nematode taxa such as Aphelenchoides, Bursaphelenchus, Ditylenchus, and Panagrolaimus. New primers and protocols are provided for successful generation of sequences useful in future investigations of nematode systematics.

The 18S small subunit (SSU) ribosomal DNA sequence is one of the most useful molecular loci for identification and phylogeny reconstruction of agriculturally important nematodes. Various pairs of universal primers have been developed in the past to amplify short and long nematode sequences. However, certain nematode taxa were not readily amplified and/or sequenced with the existing primer tools. Frequently, the center region of a roughly 1,000 nucleotide segment would be lost. Therefore new primers were developed based on a very large 276 taxon alignment of 124 agriculturally important nematode species, and tested on problematic nematode taxa such as Aphelenchoides, Bursaphelenchus, Ditylenchus, and Panagrolaimus. New primers and protocols are provided for successful generation of sequences useful in future investigations of nematode systematics.

Figure 1

Position diagram of 18S primers constructed with pDRAW32 DNA analysis by AcaClone software. http://www.acaclone.com/ Primer pairs in green and blue are older universal primers (Table 2), and those in red were generated in this study (Table 2).

Figure 2

Comparison of 18S primers by the PCR amplification. (A) G18S4 and D2AR; (B) 18S-CL-F and D2AR; (C) 18S-CL-F3 and D2AR; M: DNA ladder (0.1-4.0kb); 1: Panagrolaimus sp. Idaho; 2: Aphelenchoides bicaudatus, Maryland; 3: Bursaphelenchus sp. MX; 4: Ditylenchus sp. Idaho; NC was the negative control. The PCRs were performed with TaKaRa Ex Taq as described in Materials and Methods and the conditions: 95°C for 5 min, 35X (95°C for 30′, 50°C for 45′, 72°C for 2 min 30′), 72°C for 2 min, 4°C until finish. An approximately 2,900 nt amplicon was generated.