A new primer set for amplification of ITS-rDNA in Ditylenchus destructor

A technique was developed for the identification of Ditylenchus destructor nematode belonging to the Ditylenchus genus, based on the use of different primers for polymerase chain reaction (PCR). Two universal ribosomal primers were amplified to the internal transcribed spacer region ITS-rDNA. The sequencing of PCR products confirmed the polymorphism between species. The primers were sensitive to generate a particular band of the correct size (300bp) from the DNA template of a single, separate D. destructor stage of development. Screening populations of D. destructor from Iran and the Russian Federation have tested the reliability of the primers, and the expected size of the band was produced for all test populations. Ditylenchus destructor closely related species have also been tested and no specific band was amplified. Such results showed that the primers currently developed are useful for quantifying the D. destructor density in potato tuber.


Introduction
Since the advent of polymerase chain reaction (PCR) and a large amount of genetic data produced with DNA sequencing, molecular-based detection tools have been widely developed and successfully used for plant parasite nematodes diagnosis. Molecular detection tools have the following advantages compared to other approaches, (i) can be used in a high throughput manner, (ii) DNA information can be easily acquired with a large number of databases and sequencing information, (iii) inexpensive, fast and accurate, (iv) DNA markers are independent of phenotypic variation and nematode developmental stage [1]. DNA-based detection tools make excellent nematode diagnostic methods because they are simple, accurate and quick [2,3] and can be used with a wide variety of sample types, including host tissue, eggs, egg masses, soil extracts and fixed samples [4]. The sequences contain readily detectable genetic markers in the form of tandem repeats used to create phylogenetic trees [5] for the evaluation and diagnosis of genetically related populations [6]. Because nematodes species descriptions have historically been focused on the idea of morphological or typological organisms, molecular techniques have recently shown that many assumed monospecific species are siblings or cryptic species, genetically distinct but shared common morphological diagnostic characteristics [7][8][9]. [10, 11] and [12] used and recommended the specific primers to identify D. destructor rDNA ITS regions have also been reported to be used successfully for phylogenetic analysis [11][12][13][14]. Nonetheless, the definition of nematode species has been debated recently, indicating that species delimitation should be based primarily on an amalgamation of polyphasic taxonomy concepts, which assembles and assimilates all available data and information (phenotypic, genotypic and phylogenetic) used to delimit taxa at all levels [8,9,15]. The main accessible strategy that possibly can separate among the haplotypes is those of [12, 16,17] D. gigas have been developed [12,13,18,19]. This method is suitable for the identification of species in monospecific samples but cannot be used if the sample contains more than one nematode species using species-specific SCAR or ITS-rRNA primers [11,12,[20][21][22]. This study aimed to develop a PCR species-specific primers with sensitivity and reliability based on the sequence analysis for the molecular identification of D. destructor from other Ditylenchus species.
Materials and methods DNA extraction. Nematodes were extracted from potatoes (Solanum tuberosum L.) collected from different regions in the Russian Federation and Iran. Several nematode specimens from the population were put into a drop of water and cut by a scalpel under a camera-equipped ZEISS Axioskop50® microscope. DNA extraction from the material under this research was laid out by treating the specimens with Proteinase K that was followed by removing proteins with no extraction with organic solvents. For this purpose, a DNA-Ekstran-2 set No EX-511-100 (Synthol, Moscow) was used.
PCR with Species-specific primers. The first PCR amplification mixture (final volume 25 µl) was prepared as follows in a PCR tube (Table 1). The primer DITdesR and DITuniF were used for PCR Species-specific amplification. The contents were mixed gently by overtaxing. The reaction was performed in a thermal cycler, which involved the following stepwise procedure: denaturation of the template at 95 °C for 3 minutes, annealing at 95 °C for 35 seconds followed by extension at 63.5 °C for 30 seconds, 72 °C, 30 seconds and 5 minutes at 72 °C for 35 cycles. Negative control was included with each set of amplification. Then, The PCR products were subsequently partitioned according to their size on 1 % agarose gel electrophoresis and visualized by Gel documentation or purified for sequencing.
Sequencing and Phylogenetic analysis. The amplified PCR product obtained after purification by Thermo Scientific Gene JET Gel Extraction Kit was sequenced by Sanger's dideoxy cycle by Genetic Analyzer AB-3500 (Applied Biosystems, USA). Primitive comparison of sequencing results with the GeneBank genetic sequence database was done by the NCBI BLAST web site (http://www.ncbi.nlm.nih.gov/BLAST). The results are presented in percentage values, the DNA sequence available in the GeneBank homologous to those examined were analyzed along with the newly sequenced one.
Design of Species-specific Primers. The ITS sequences of D. destructor including MN122076, MN307126, MN307128, MN493767, MN658597, MN658599, MN658637, MN658638 and D. dipsaci: MG676655, MG676656, MG676656, D. gigas: KJ653270, KJ653267 which were retrieved from NCBI and were used for the design of specific primers ( Table 2). The specific forward and reverse primers were designed from the ITS regions using Primer Premier DITdesR and DITuniF to generate an expected fragment of about 126 bp in length and verified using BLAST (http://www.ncbi.nlm.nih.gov/blast) to exclude nonspecific reactions with other closely related species.  (Table 4). The sequence analysis revealed sequence variability between different geographical populations of D. destructor isolated from different host plants.  Design of Species-specific Primers. A first primer named dsn.1 (Table 5) was designed to have some nucleotides mismatches observed when comparing D. destructor, D. dipsaci, and D. gigas (Fig 1, 2). At present, a set of species-specific primers for D. destructor were developed based on the sequence differences in the rDNA-ITS region of D. destructor. The PCR amplification by the species-specific primers demonstrated that it could amplify a single, stable and clear band for a single adult and different geographical populations of D. destructor. The specificity and reliability of the primers were also demonstrated in vitro conditions. Given the specificity, sensitivity, and reliability of the primers for D. destructor, the diagnostic primers could provide a rapid and reliable molecular marker for identification or detection of D. destructor. Typically, testing diagnostic primers usually need more samples with similar morphological characteristics and more geographically disparate locations [23]. Possibly, the species-specific primers designed in the present work may cross-react with other species in the genus of Ditylenchus and mismatches at the primer-binding site to produce the same specific band.   Test of Species-specific Primers. To evaluate the sensitivity of the species-specific primers, the PCR amplified products for different numbers of D. destructor amplified using the primers (dsn.1 and dsn.2) were shown in (Fig 3). A single band with a length of 300 bp was obtained from DNA templates extracted D. destructor. The method proved suitable for D. destructor sensitive identification of DNA samples. The specificity and reliability of the specific primers were confirmed by yielding the expected fragment sizes (300 bp) for all the populations of D. destructor and no products were detected for the tested of the species D. dipsaci and D. gigas (Fig 4).

Conclusions
In this study, a set of species-specific primers (named dsn.1 /dsn.2) was designed firstly for molecular identification of D. destructor based on the sequence analysis of rDNA-ITS. The specificity, sensitivity, and reliability of the primers were repeatedly ЗАЩИТА РАСТЕНИЙ demonstrated. Therefore, the developed specific primers should be a rapid and accurate molecular protocol for the diagnosis of D. destructor and also be fundamental for effective management of the nematode.