MATERIALS AND METHODS

Survey of certain cereal aphid species: Field survey of aphid species was carried out during the period 2008-2009 in different localities of Egypt. Eleven cereal aphid species were collected from different localities allover the years. The species are listed alphabetically by scientific name in Table 1.

Samples from the infested plants were transferred to the laboratory and alate individuals of aphid were mounted on slides for identification by using available keys. Identification of these species was conducted according to the key of Fathi and El-Fatih (2009). The eleven species were reared on their host separately, where one apterous adult was transferred by a brush to a healthy plant. Nymphs were leaved to feed and developed to adults. This method was repeated for two off-springs. The adults of the second offspring of each species were put in tubes and preserved at -20°C until use in the biochemical and molecular studies.

RAPD-PCR analysis
DNA extraction: DNA were extracted from eleven different species of adult Aphid.Animal tissues were ground under liquid nitrogen to a fine powder, then bulked DNA extraction was performed using DNeasy plant Mini Kit (QIAGEN).

Table 1:	List of eleven cereal aphid species with their host plants and localities in Egypt

Polymerase Chain Reaction (PCR): PCR amplification was performed using eight random 10 mer arbitrary primers (Table 2) these primers synthesized by (Operon biotechnologies, Inc. Germany).

Amplification was conducted in 25 μL reaction volume containing the following reagents: 2.5 μL of dNTPs (2.5 mM), 2.5 μL MgCl₂ (2.5 mM), and 2.5 μL of 10 x buffer, 3.0 μL of primer (10 pmol), 3.0 μL of template DNA (25 ng μL^-1), 1 μL of Taq polymerase (1U μL^-1) and 10.5 μL of sterile dd H₂O. The DNA amplifications were performed in an automated thermal cycle (model Techno 512) programmed for one cycle at 94°C for 4 min followed by 45 cycles of 1 min at 94°C, 1 min at 36°C, and 2 min at 72°C. the reaction was finally stored at 72°C for 10 min. Amplified products were size-fractioned using ladder marker (100 bp+1.5 Kbp) by electrophoresis in 1.5% agarose gels in TBE buffer at 120 V for 1 h. the bands were visualized by ethidium bromide under UV florescence and photographed.

ISSR-PCR analysis
DNA extraction: Eleven different species of adult Aphids samples were collected and extracted DNA from them. Animal tissues were ground under liquid nitrogen to a fine powder, and then bulked DNA extraction was performed using DNeasy plant Mini Kit (QIAGEN).

Polymerase Chain Reaction (PCR): PCR amplification was performed using five Inter Simple Sequence Repeat (ISSR) Table 3.

Amplification was conducted in 25 μL reaction volume containing the following reagents: 2.5 μL of dNTPs (2.5 mM), 2.5 μL MgCl₂ (2.5 mM), and 2.5 μL of 10 x buffer, 3.0 μL of primer (10 pmol), 3.0 μL of template DNA (25 ng μL^-1), 1 μL of Taq polymerase (1U μL^-1) and 10.5 μL of sterile dd H₂O. The DNA amplifications were performed in an automated thermal cycle (model Techno 512) The PCRs were programmed for one cycle at 94°C for 4 min followed by 45 cycles of 1 min at 94°C, 1 min at 57°C and 2 min at 72°C. The reaction was finally stored at 72°C for 10 min. Amplified products were size-fractioned using ladder marker100bp (1000, 900, 800, 700, 60, 500, 400, 300, 200 and 100 bp) by electrophoresis in 1.5% agarose gels in TBE buffer at 120 V for 30 min the bands were visualized by ethidium bromide under UV florescence and photographed.

Densitometry scanning and analysis: All gels resulted from DNA fingerprints, were scanned using Bio-Rad GelDoc2000 to calculate the pair-wise differences matrix and plot the dendrogram among different aphid species.

Table 2:	List of the RAPD primers and their nucleotide sequences

Table 3:	List of the ISSRs primers and their nucleotide sequences

Table 4:	Profile of diagnostic morphological characters, to identify the eleven aphid species, under consideration, expressed as zero and one values

Phylogenetic relationship among the eleven species: Genetic similarities and genetic relatedness among the eleven cereal aphid species were based on data obtained of three different criteria; eight RAPD primers and five ISSRs primers as molecular markers as well as twenty-six diagnostic morphological characters. These data were subjected to using SPSS computer program to support the existence of high level of genetic relatedness among the eleven cereal aphid species. The relatedness dendrogram was indicated two main clusters. The first cluster included ten aphid species while, the second included only S. scirpus with similarity matrix percentage of 73%. The first cluster was divided into two sub-clusters with similarity matrix percentage of 82%; the first one included six aphid species with similarity matrix percentage of 88%; T. africana, A. corni, S. avenae, M. dirhodum, R. maidis and R. padi. While the second sub-cluster contained four aphid species with similarity matrix percentage of 84%; S. rotundiventrus, S. minuta and S. graminum and H. pruni. The highest similarity matrix percentage in this phylogenetic relationship was 98% between T. Africana and A. corni, while the lowest similarity matrix percentage found between S. scirpus and T. africana (73%) (Fig. 3). These results indicated the success of these techniques with morphological characters to draw the phylogenetic relationship of these aphids' species whereas the species those belonging to the same genus are closest to each other.

RESULTS

Random amplified polymorphic DNA (RAPD): Eight RAPD primers were tested against the eleven cereal aphid species to find markers for identification of these species (Fig. 1). These primers generated 167 fragments, 97 bands of them were considered as markers for different species (58.1%), the highest number of markers was 20 bands generated by OPC-01 and OPC-13, while the lowest number of markers was 4 bands generated by OPD-07. the highest number of markers was 12 bands detected for S. graminum, while the lowest number of markers was 5 bands detected for M. dirhodum (Table 5). Thirty-seven common bands (22.2%) were detected among the eleven species found by the eight tested primers.

OPC-01 primer: This primer generated 23 bands with molecular weight ranged from 903 to 111 bp. and the average number of bands generated in different species ranged from 8 bands in S. avenae and 4 bands in M. dirhodum. This primer showed 87.1% polymorphism whereas three common bands among the eleven species were detected (784, 378 and 250 bp.). Twenty marker bands were detected for the eleven species; the highest number of markers was three bands for S. graminum and S avenae, while the lowest number was one band in R. maidis, S. minuta, M. dirhodum and H. pruni. While the other five species, each of them has two marker bands. OPC-13 primer: This primer generated 30 bands with molecular weight ranged from 1360 to 105 bp. and the average number of bands generated in different species ranged from 11 bands in H.pruni and 4 bands R. maidis. This primer showed 90% polymorphism whereas three common bands were detected among the eleven species with molecular weights 764, 431 and 355 bp. Twenty marker bands were detected for the eleven species, the highest number of markers was five bands for H. pruni, while the lowest number was one band for six species; A. corni, T. africana, R. maidis, S. graminum, S. avenae and S. scirpus. Three marker bands were detected for two species; S. rotundiventrus and S. minuta, two marker bands for R. padi and M. dirhodum.

Fig. 1:	RAPD banding patterns of eleven cereal aphid species generated by eight random primers. M, 1500 bp marker; 1, Anoecia corni; 2, Tetrenura Africana; 3, Rhopalosiphum maidis; 4, Rhopalosiphum padi; 5, Schizaphis graminum; 6, Schizaphis rotundiventrus; 7, Schizaphis minuta; 8, Sitobion avaenae; 9, Metopolophum dirhodum; 10, Hyalopterous pruni; 11, Saltusaphis scirpus

PD-07 primer: This primer generated 16 bands with molecular weight ranged from 1231 to 223 bp. and the average number of bands generated in different species ranged from 10 bands in six species; R. maidis, S. rotundiventrus, S. avenae, M. dirhodum, H. pruni and S. scirpus to 5 bands in S. graminum and S. minuta. This primer showed 68.75% polymorphism whereas five common bands (1231, 750, 520, 392 and 328 bp) among the eleven species were detected. Only four marker bands; 886, 639, 795 and 935 bp were detected for A. corni, R. maidis, R. padi and M. dirhodum, respectively.

OPE-03 primer: This primer generated 24 bands with molecular weight ranged from 1186 to 208 bp. and the average number of bands generated in different species ranged from 12 bands in A. corni to 5 bands in S. rotundiventrus. This primer showed 83.3% polymorphism whereas four common bands (737, 387, 356 and 208 bp) among the eleven species were detected. Thirteen marker bands were detected for eight species, three marker bands for S. scirpus; two marker bands for three species; A. corni, T. africana and S. avenae. While only one marker band was detected for four species; R. maidis, R. padi, S. minuta and H. pruni.

OPE-06 primer: This primer generated 19 bands with molecular weight ranged from 1132 to 190 bp. and the average number of bands generated in different species ranged from 8 bands in A. corni to 4 bands in S. graminum. This primer showed 79% polymorphism whereas four common bands ( 421, 332, 222 and 190 bp ) among the eleven species were detected. Ten marker bands were detected for seven species, two marker bands for three species; A. corni, S. minuta and S. scripus. While one marker band (monomorphic) was detected for four species; R. padi, S. rotundiventrus, S. avenae and H. pruni.

OPI-17 primer: This primer generated 21 bands with molecular weight ranged from 1343 to 162 bp and the average number of bands generated in different species ranged from 10 bands in T. africana and S. graminum to 7 bands in two species; A. corni and R. padi. This primer showed 66.7% polymorphism whereas seven common bands (988, 841, 656, 557, 486, 419 and 343 bp.) among the eleven species were detected. fourteen marker bands (monomorphic) were detected for nine species, the highest number of markers was five bands detected for S. graminum, while the lowest number was one band was detected for seven; A. corni, R. maidis, S. minuta, S. rotundiventrus, S. avenae, M. dirhodum and H. pruni. While two marker bands were detected for T. africana. No marker bands for two species; R. padi and S. scirpus.

OPL-20 primer: This prime generated 15 different fragments with molecular weight ranged from 1240 to 202 bp the average number of bands generated in different species ranged from 9 bands in R. maidis to 7 bands in S. rotundiventrus, H. pruni and S. scirpus; while the other eight species have 8 bands. This primer showed 60% polymorphism whereas, six common bands were detected by this primer with molecular weights 1240, 731, 662, 507, 400 and 202 bp. Six marker bands in six different species were detected (one marker band for each species); A. corni, T. africana, R. maidis, R. padi, S. rotundiventrus and S. avenae. No marker bands for the other six species.

OPQ-15 primer: This primer generated 19 bands with molecular weight ranged from 1162 to 202 bp. and the average number of bands generated in different species ranged from 8 bands in three species; T. african, R. maidis, R. padi, to 5 bands in M. dirhodum. This primer showed 73.7% polymorphism whereas five common bands among the eleven species were detected (683, 410, 341, 238 and 202 bp). Ten marker bands (monomorphic) were detected for nine species; the highest number of markers was two bands detected for S. graminum. While just one marker band was detected for eight species; A. corni, T. africana, R. maidis, R. padi, S. rotundiventrus, S. minuta, H. pruni and S. scirpus. While no marker bands were detected for S. avenae and M. dirhodium.

Inter simple sequence repeats (ISSRs): Five ISSRs primers were tested against the eleven cereal aphid species to find markers for identification of these species (Fig. 2). These primers generated 122 fragments, 69 bands of these fragments were considered as markers for different species (56.6%), the highest number of markers was 15 bands generated by two primers; HP-09 and HP-13, while the lowest number of markers was 12 bands generated by HP-14 primer. A. corni had the highest number of marker bands (10 bands), while the lowest number of markers was 3 bands detected for S. scirpus (Table 6). Twenty-one common bands (17.2%) were detected among the eleven species by these five tested primers.

HP-09 primer: This primer generated 23 bands with molecular weight ranged from 1109 to 117 bp. and the average number of bands generated in different species ranged from 8 bands in A. corni to 5 bands in R. padi and S. avenae. This primer showed 82.6% polymorphism whereas 4 common bands (422, 349, 254 and 172 bp) were detected among the eleven species. Fifteen marker bands (monomorphic) were detected for ten species; the highest number of markers was three bands were detected for A. corni, while the lowest number was one marker band was detected for six species; R. padi, S. graminum, S. minuta, S. avenae, H. pruni and S. scirpus. While no marker bands were detected for M. dirhodium.

HP-11 primer: This primer generated 30 bands with molecular weight ranged from 1301 to 124 bp. This primer showed 73.3% polymorphism, where eight common bands (930, 780, 685, 516, 436, 288, 242 and 174 bp) were detected among the eleven species. The average number of bands generated in different species ranged from 15 bands in S. rotundiventrus to 10 bands in S. scirpus. Fourteen marker bands were detected for the eleven species; the highest number of markers was two bands were detected for three species; A. corni, S. graminum and S. rotundiventrus, while the lowest number was one marker band was detected for the other eight species.

HP-12 primer: This primer generated 22 bands with molecular weight ranged from 842 to 95 bp. This primer showed 90.9% polymorphism, where two common bands (323 and 230 bp) were detected among the eleven species. The average number of bands generated in different species ranged from 9 bands in S. avenae to 2 bands in T. africana. Thirteen marker bands were detected for eight species; the highest number of markers was three bands were detected for S. avenae, while the lowest number was one marker band was detected for four species; A. corni, R. maidis, R. padi and S. rotundiventrus. Two marker bands were detected for three species; S. graminum, M. dirodium and H. pruni. While no marker bands were detected in T. africana, S. minuta and S. scirpus.

HP-13 primer: This primer generated 25 bands with molecular weight ranged from 1016 to 123 bp. This primer showed 88% polymorphism, where three common bands (640, 398 and 174 bp) were detected among the eleven species. The average number of bands generated in different species ranged from 10 bands in S. avenae to 5 bands in H. pruni. Fifteen marker bands were detected for the eleven species; the highest number of markers was two bands were detected for four species; A. corni, T. africana, R. maidis and S. graminum. While the lowest number was one marker band was detected for the other seven species.

Table 5:	Molecular markers generated by eight RAPD primers for eleven cereal aphid species in Egypt

HP-14 primer: This primer generated 22 bands with molecular weight ranged from 963 to 32 bp. This primer showed 81.8% polymorphism, where four common bands (261, 192, 153 and 101 bp) were detected among the eleven species. The average number of bands generated in different species ranged from 8 bands in S. avenae, H. pruni and S. scirpus to 5 bands in S. minuta and M. dirhodium. Twelve marker bands were detected for nine species; the highest number of markers was two bands were detected for three species; A. corni, S. rotundiventrus and S. avenae. While the lowest number was one marker band was detected for six species; T. africana, R. maidis, R. padi, S. graminum, S. minuta and M. dirhodium. While no marker bands were detected for two species; H. pruni and S. scirpus.

Phylogenetic relationship among the eleven species: Genetic similarities and genetic relatedness amongst the eleven cereal aphid species were based on data obtained of three different criteria; eight RAPD primers and five ISSRs primers as molecular markers and as well as twenty-three diagnostic morphological characters. These data were subjected to using SPSS computer program to support the existence of high level of genetic relatedness amongst the eleven cereal aphid species. The relatedness dendrogram was indicated two main clusters. The first cluster included ten aphid species while, the second included A. corni only with similarity matrix percentage of 73%. The first cluster was divided into two sub-clusters with similarity matrix percentage of 82%; the first one included six aphid species with similarity matrix percentage of 88%; H. pruni, S. scirpus, S. avenae, M. dirhodum, S. rotundiventrus and S. minuta.

Fig. 2:	ISSR banding patterns of eleven cereal aphid species generated by five primers. M, 1000 bp marker; 1: Anoecia corni, 2: Tetrenura Africana; 3: Rhopalosiphum maidis; 4: Rhopalosiphum padi; 5: Schizaphis graminum; 6: Schizaphis rotundiventrus; 7: Schizaphis minuta; 8: Sitobion avaenae; 9: Metopolophum dirhodum; 10: Hyalopterous pruni; 11: Saltusaphis scirpus

While the second sub-cluster contained four aphid species with similarity matrix percentage of 84%; S. graminum, R. maidis, R. padi and T. africana. The highest similarity matrix percentage in this phylogenetic relationship was 98% between H. pruni and S. scirpus, while the lowest similarity matrix percentage found between A. corni and H. pruni (73%) (Fig. 3). These results indicated the success of these techniques with morphological characters to draw the phylogenetic relationship of these aphids’ species whereas the species those belonging to the same genus are closest to each other.

DISCUSSION

Some of the insect species are easy to identify and categorize, while for others, such as aphids species are difficult because of their small size and morphological similarity. Moreover, it is further difficult to identify morphological variation due toenvironmental factors by available traditional methods (Loxdale and Brookes, 1989; Cenis et al., 1993; Figueroa et al., 1999). To overcome these problems, the advanced molecular techniques, viz., randomly amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) and simple sequence repeats (ISSRs) have been useful tools in assessing insect genetic diversity (Black et al., 1992; Cenis, 2003; Gobbi et al., 2003; Sartor et al., 2008; Sharma et al., 2008; Perumal et al., 2009; Qiu et al., 2009).

Table 6:	Molecular markers generated by five ISSRs primers for eleven cereal aphid species in Egypt

Fig. 3:	Phylogenetic dendrogram among eleven cereal aphid species based on three criteria; RAPD markers, ISSRs markers and certain diagnostic morphological characters

RAPD markers have become the most common yardsticks for measuring genetic differences between individuals, within and between related species or population (Jain et al., 2010). This technique also used to distinguish different geographical and/or host associated populations of some cryptic complex species (Zitoudi et al., 2001; Bulman et al., 2005; Lopes-da- Silva and Vieira, 2007; Helmi, 2011).

Inter simple sequence repeats (ISSRs) is a valuable addition to the inventory of PCR-based methods for rapid, large-scale screening of genetic variations. The vagaries of PCR and the chosen method of band detection limit any PCR-based marker (Wolfe and Liston, 1998). But ISSRs markers are typically highly reproducible, due to stringent annealing temperatures, long primers, and low primer-template mismatch (that is, the primers are not ‘arbitrary’, but designed a priori to anchor onto anonymous SSR loci; Wolfe et al., 1998) and ISSRs can reveal polymorphisms without more elaborate detection protocols (Esselman et al., 1999).

ISSRs method has shown much promise for the study of the population biology of plants (Provan et al., 1996; Wolff and Morgan-Richards, 1998; Parsons et al., 1997; Clausing et al., 2000; Hess et al., 2000; McGregor et al., 2000), but rarely used in animals (Reddy et al., 1999; Kostia et al., 2000; Abbot, 2001). There are two available known studies in use of ISSRs in aphids; one of them was on population-level studies in two species of cyclically parthenogenetic aphids; Acyrthosiphon pisum and Pemphigus obesinymphae (Abbot et al., 2001) who reported that ISSRs are suitable for invertebrate populations those have small size bodies and low levels of within-population variation. While the other study was on host-associated genetic differences and regional differences among the green bug, Schizaphis graminum biotypes (Weng et al., 2007) who cited that the use of ISSRs would be useful for aphid genetic, ecological, and evolutionary studies and can potentially shorten the time and cost for biotype identification.

CONCLUSION

Molecular fingerprinting of eleven cereal aphid species collected from Egypt were carried out using two modern genetic techniques; RAPD-PCR and ISSRs. These techniques successfully generated many molecular markers for different studied species, those could be used to identify these aphid species and differentiate among them. Also these two techniques in addition to some diagnostic morphological characters were used to determine the Phylogenetic relationship among the eleven aphid species.