Background

Entomopathogenic nematodes (EPNs) are a promising candidate for biological control of insects due to their ability to find hosts, integrity to non-target organisms and the environment, high reproductive potential, capacity to be mass-produced and to be used with other agrochemical pesticides and fertilizers (Laznik and Trdan 2014). Two EPN families, Heterorhabditidae and Steinernematidae, have separately evolved collaborative associations with insect pathogenic bacteria (Photorhabdus and Xenorhabdus) (Adams et al. 2006). Morphological characterizations cannot be used unambiguously to rank new isolates into a particular species; the tendency of using these nematodes as biological control agents depends upon the appliance required for rapid and accurate methods to determine the genetic diversity among existent populations of ENPs species (Hashmi et al. 1996). Molecular markers have been used to study genetic diversity; it mainly exposes naturally occurring polymorphisms at DNA level that makes it a powerful tool to determine the effectiveness of various factors on genetic diversity and population structure (Li et al. 2014). DNA markers are numerous and separate and are widespread throughout the genome. They are highly polymorphic and stable and allow organism identification in each developmental stage (Agarwal et al. 2008). Random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), simple sequence repeats (SSR), inter-simple sequence repeats (ISSR), single-strand conformation polymorphism (SSCP), and sequence-related amplified polymorphism (SRAP) are highly polymorphic and constant and have been widely employed in molecular taxonomy, gene cloning, phylogeny, and genetic mapping, (Gasser et al. 2006; Mutlu et al. 2008; Zhao et al. 2009). RAPD has been used for the genetic variability and phylogenetic studies of different organisms including insects, EPNs, animals, and plants (Moghaieb et al. 2004). RAPD analysis was used for the characterizations of a new species of Heterorhabditis from Hawaii (Gardner et al. 1994). SRAP is known as a useful molecular marker system based on its reproducibility and low cost (Li and Quiros 2001). Currently, SRAP has been successfully utilized to evaluate genetic diversity in species. It has been shown to be more instructive than other PCR-based techniques in discovering genetic diversity (Budak et al. 2004) and has been used to study the genetic diversity and relationships among several species (Abedian et al. 2012).

As there is no data about the application of SRAP analysis in EPN identification and taxonomy, this study aimed to compare the accuracy of SRAP and RAPD analyses in detecting the genetic variations among seven EPN species from Steinernematidae.

Materials and methods

Nematodes culture

Seven steinernematid species were used, and their names and geographical origins are listed in Table 1.

Table 1 Names of seven steinernematid species and their geographical origin
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According to the method of Dutky et al. (1964), the EPNs were cultured, using the last instar larvae of the greater wax moth (Galleria mellonella L.) as a host.

Extraction of DNA

Total genomic DNA was extracted according to the method of Kary et al. (2009) as follows:

The nematode (5000 IJ) were crushed in 15 μl 1 × PCR buffer and transferred to a sterilized 0.2-ml tube containing 10 μl of the same buffer. The tube was incubated at − 70 °C for 15 min and thawed at 60 °C then inoculated with 2 μl of 60 μg ml−1 proteinase K. The tube was incubated at 65 °C for 2 h and then heated at 95 °C for 15 min. After centrifugation at 16.000 rpm for 15 min., the supernatant including nematode DNA was collected and stored at − 70 °C until use. The quality and quantity of DNA were determined using 0.8% agarose gel electrophoresis and spectrophotometer.

RAPD analysis

PCR analysis

PCR reactions mixture containing 200 μM dNTPs, 1 μM of 9 arbitrary 10-mer primers (Operon Technology, Inc., Alameda, CA, USA), 10 ng DNA, 0.5 units of Red Hot Taq polymerase (AB gene House, UK) and 10 × Taq polymerase buffer (AB gene House, UK), and total volume are adjusted to 20 μl. For DNA amplification, Biometra thermal cycler (2720) was programmed as follows: 94 °C for 5 min, followed by 35 cycles of 94 °C for 1 min, 35 °C for 1 min, 72 °C for 1 min, and 72 °C for 7 min. The products were analyzed by electrophoresis in 1% agarose in TAE buffer, stained by ethidium bromide, and photographed under UV light. The sequence of the tested primers is listed in Table 2.

Table 2 Names and sequences of RAPD primers used to assess the genetic variability among the seven steinernematid species
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SRAP analysis

PCR reactions were performed in a total volume of 20 μl containing 2 μl of the DNA suspension, 2 μl of 10 × PCR buffer, 2 μl of MgCl2 (25 mM), 1 μl of dNTP mixture (10 mM of each dNTP), 0.5 U of Taq DNA polymerase, 10.5 μl of dd H2O, and 1 μl of each primers. PCR program includes 5 min of initial denaturation at 94 °C. The first 5 cycles were run at 94 °C, 1 min; 35 °C, 1 min; and 72 °C, 1 min, for denaturing, annealing, and extension, respectively. Then, the annealing temperature was raised to 50 °C for another 35 cycles (Li and Quiros 2001), and the sequence of the primers is listed in Table 3.

Table 3 Names and sequences of SRAP primers used to assess the genetic variability among the seven steinernematid species
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Band scoring and cluster analysis

Gel Doc 2000 Bio-Rad system was used for RAPD and SRAP, and gel image scanning and One Software v 4.0.1 (Bio-Rad Laboratories, Hercules, Co., USA) were used for analyses with quantity. To calculate the pairwise differences matrix and plot the dendrogram among the seven EPN species, the Systat ver. 7 computer program was used. Cluster analysis based on similarity matrices obtained with the unweighted pair group method using the arithmetic average (UPGMA) to estimate the dendrogram.

Results and discussion

RAPD markers were used to study genetic diversity and population dynamics of various isolates in Heterorhabditidae and Steinernematidae (Hashmi and Gaugler 1997; Moghaieb et al. 2004). Nine random RAPD primers were used to evaluate the genetic polymorphism among the seven EPN species which generated 65 bands, 89 (73%) of them were polymorphic. The fragment size ranged between 110 and 1500 bp approximately, and the OPE-A-07 primer scored the highest number of RAPD bands (14 bands) while OPE-D-20 scored the lowest band number (7 bands) (Fig. 1, Table 4). Table 5 shows the specific RAPD marker for different species, and two specific markers were recorded for S. abbasi 1, S. riobrave, and S. glaseri followed by S. carpocapsae DD 136 (one specific marker), while none for S. abbasi 2, S. carpocapsae all, and S. scapterisci.

Fig. 1
figure 1

Genetic polymorphism among seven species belongs to the family Steinernematidae as revealed by RAPD analysis. M, 1 kbp plus DNA ladder; 1–7, the nematode species (1, S. abbasi 1; 2, S. abbasi 2; 3, S. carpocapsae DD 136; 4, S. carpocapsae all; 5, S. riobrave; 6, S. scapterisci; 7, S. glaseri)

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Table 4 Total number of scorable bands, polymorphism percentage, and band size of RAPD markers obtained by nine primers
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Table 5 Steinernematid species and their specific RAPD markers
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RAPD has been used to explore the genetic diversity among species and strains of EPNs (Hashmi and Gaugler 1998), to define phylogenetic relationships (Liu and Berry 1996), and complement descriptions of new species (Stock et al. 1996). Padmanaban et al. (2014) developed RAPD markers to discriminate the genetic diversity in 25 recovered EPNs (Steinernema spp. and Heterorhabditis spp.) and found that polymorphism percentage that recorded in all the strains was 12.31%. The obtained data agrees with Ibrahim (2009), and by using RAPD analysis, the genetic polymorphism reached 96.9% and 91.4% between the nematode species belonging to Steinernema and Heterorhabditis, respectively. The genetic diversity among the EPN species from the genus Steinernema showed a high resemblance of shared DNA fragments between S. riobrave and S. scapterisci (95.4%) followed by S. scapterisci and S. carpocapsae all (89.4%). Hashmi et al. (1996) obtained similar data, where they screened 80 arbitrary 10-mer random primers for determining genetic variability among isolates of Heterorhabditis. Cluster analysis for Steinernema species showed that the cluster was divided into two branches, the first one was S. feltiae and the second was subdivided into two branches, one had S. carpocapsae and the other had S. glaseri. Shapiro et al. (1997) used RAPD-PCR to compare genetic variation in two strains of Heterorhabditis bacteriophora and had found a similar genetic heterogeneity of isolated population of H. bacteriophora to that reared in the laboratory for a long period. Moghaieb et al. (2004) employed protein banding pattern (SDS-PAGE) and RAPD markers to discriminate between two heat tolerant EPN isolates (HTS1 and HTS 2) and their original strain ISK-2, and nine arbitrary 10-mer primers were used. A total of 5 (21.7%) and 18 (39.1%) polymorphic bands were found out for protein and RAPD, respectively. They found that the EPN-specific markers represent 14% of the total markers detected by both analyses and 70% of them were RAPD markers, and these markers are recommended in providing a quick and reliable discrimination technique, compared to the protein analyses, to identify and distinguish between the heat tolerance and other EPN isolates.

SRAP is known as a useful molecular marker system, based on its propagation, low cost, and little knowledge that is necessary for target sequences in a species (Li and Quiros, 2001). The distinguishing power of molecular markers has been arranged as SRAP > SSR > ISSR > RAPD (Budak et al. 2004).

In this study, 12 different SRAP primer pairs among seven species of tested EPN were used. The data revealed that 97 bands out of 107 were polymorphic (90.6%) (Fig. 2, Table 6). The Me3-em2 primer recorded the highest number of bands (13 bands) followed by Me2-em3 (11 bands) while Me3-em3 and Me2-em2 recorded the lowest band number (6 bands). The genotype-specific SRAP markers for the different EPN species are listed in Table 7. The highest number of SRAP specific markers was scored for S. glaseri (7 markers), then 4 markers for S. abbasi 2, followed by S. riobrave (3 markers), one marker for S. abbasi 1 while none for S. carpocapsae DD 136, S. carpocapsae all, and S. scapterisci. Devran and Baysal (2012) used 20 SRAP primer combinations to evaluate the genetic variations in M. incognita isolates. They found that the highest number of polymorphic bands was produced by Me5-em4 SRAP primer combination, while the lowest number of polymorphic bands was obtained by Me1-em1 SRAP primer combination.

Fig. 2
figure 2

The genetic variability among nematode species as revealed by SRAP analysis, M, 1 kbp plus DNA ladder, 1–7, the nematode species (1, S. abbasi 1; 2, S. abbasi 2; 3, S. carpocapsae DD 136; 4, S. carpocapsae all; 5, S. riobrave; 6, S. scapterisci; 7, S. glaseri)

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Table 6 Total number of scorable bands, polymorphism percentage, and band size of SRAP markers obtained by 12 primers
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Table 7 Steinernematid species and their specific SRAP markers
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The relationships among seven species of EPN

The obtained data from RAPD markers were used to calculate the phylogenetic tree which revealed the genetic relationship among different species of EPN. The cluster dendrogram showed two major clusters (Fig. 3). The first cluster was divided into two branches, the first one had S. abbasi 1, and the second branch had S. abbasi 2. The second cluster was subdivided into two branches, the first one was S. glaseri and the other branch was divided into two sub-cluster, the first contained S. carpocapsae DD 136 and the second contained S. carpocapsae all, S. riobrave, and S. scapterisci.

Fig. 3
figure 3

Phylogenetic tree of seven steinernematid species based on pooled RAPD markers

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The phylogenetic tree from SRAP markers showed two major clusters as RAPD phylogenetic tree (Fig. 4). The first cluster was divided into two branches, the first one has S. abbasi 1 and the second branch had S. abbasi 2. The second cluster was subdivided into two branches, the first one is S. glaseri and the other branch was divided into two sub-clusters, the first contained S. riobrave and S. scapterisci and the second contained S. carpocapsae DD 136 and S. carpocapsae all. Therefore, the two molecular markers can be used to differentiate among the seven species of EPN.

Fig. 4
figure 4

Phylogenetic tree of seven steinernematid species based on pooled SRAP markers

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Similar results were reported by Dillman et al. (2015) who sequenced and accumulated the genomes of five Steinernema spp. (S. carpocapsae, S. feltiae, S. glaseri, S. monticolum, S. scapterisci) and supposed the relationships among the five Steinernema. S. carpocapsae and S. scapterisci had the same cluster and they were more closely to S. feltiae than S. glaseri whereas in the present study, S. carpocapsae was near to S. glaseri than S. scapterisci.

Conclusion

The present study indicated that RAPD and SRAP can be successfully used as molecular markers for estimating the genetic variability and the genetic relationships among the EPNs. Specific molecular markers can be used as specific probes for identification of different species and isolates, which can be used in biological control programs.