Molecular genetic identification of two bracon species based on RAPD-PCR and 16 S rRNA genes

Random amplified polymorphic DNA (RAPD)-PCR genomic fingerprinting and partial sequencing of the 16S rRNA gene were evaluated on two insects collected from the Egyptian field which could belong to B. hebetor and B. brevicornis to investigate their genetic relatedness and to establish the value of techniques for their identification. Nearly identical RAPD-PCR profiles and identical 467 bp fragments of the 16S rRNA genes indicated many of genetic diversity between the two insects under study. The low levels of similarity (78.21% in the partial 16S rRNA genes and 86% in RAPD-PCR) appeared between the insects B. hebetor egypt and B. brevicornis egypt. However, 16S rRNA genes and RAPD-PCR provided an effective means of differentiating between members of the taxa. Moreover, a phylogenetic tree constructed from 16S rDNA sequences showed that B. hebetor egypt clustered with the B. hebetor with a degree of similarity 92%, but B. brevicornis Egypt clustered in a separated group. However, RAPD-PCR and partial sequencing of the 16S rDNA analysis raises questions about the taxonomic positioning of the two insects isolated from the Egyptian environment.


INTRODUCTION
The identification and the use of correct parasitoids species is very important step in biological control programs to be released in the field.The identification of these parasitoids is difficult due to the most parasitoids are small, even minute, and evolving rapidly.Related species often have few or no known sufficiently invariant distinguishing morphological characters for reliable discrimination (Pungerl, 1986;Landry et al., 1993;Pinto et al., 1993;Demichelis & Manino, 1998;Kimani-Njogu et al., 1998;Stouthamer et al., 1999;Barnay et al., 2001;Chang et al., 2001) and their identification at species level depends mainly on male genitalia.Failure or delay the definition of parasite affect the outcome of the control of pest where the success of biological control programs often depends on correct identification of natural enemies (DeBach and Rosen, 1991).Today's technology allows us to identify any living thing by using a single cell.
The classification of species based on morphological features has problems because morphological attributes could change by environment (Shouche and Patole, 2000).Many molecular techniques allow ecologists and biologists to determine the genetics array of a wide variety of closely related individuals (Wolf and Rijn, 1993).Among these techniques are DNA sequencing, restriction fragment length polymorphisms (RFLP), microsatellites analysis and random amplified polymorphic DNA (RAPD) (Mulcahy et al., 1993).Bracon spp is minute and indistinguishable morphologically, further, the environmental factors influence significantly its morphology and physiology.So, identification of the wasp is problematic and its systematic needs to be clarified (Pinto, 1998).

Collection of samples:
Two spices of Bracon insects presented in the Egyptian fields.They may be Bracon.hebetor and Bracon.brevicornis and they individuals were kept in liquid nitrogen until use.

DNA extraction
Total genomic DNA was extracted from fifteen bulk of the two individual species by using GeneJET TM Genomic DNA Purification kit (Fermentas).DNA samples were diluted by TE-buffer to final concentration of 50 ng/µl to be used in polymerase chain reaction (PCR) and stored at -20°C.

PCR conditions and purification of PCR products
For identification of two Bracon species, we used two categories, RAPD-PCR and sequencing a part of mt16S rRNA genes.In RAPD-PCR method, twenty primers (table 1), obtained from Pharmacia Biotech.

Sequencing
For sequencing of 16S rDNA fragment (~467bp) we used sequencing unit which is equipped with a Tecan robot installed on a platform Genesis Workstation 150 (capillary electrophoresis) and performs the reactions are then analyzed via the sequencer 3100 Genetic Analyser (Applied Biosystems).DNA fragments to be sequenced are prepared in a final reaction concentration 6μl content 30 ng of PCR products and 5 pmol of the 16sWb or 16s.Sh primers.Sequence analysis was done with the Sequencher 3.0 software.

Data analysis
Data of reducible RAPD markers were scored "1" or "0" for each sample "1" was assigned for the presence of a band and "0" for its absence.These data were used in counting the number of total amplified markers in two Bracon spp.Moreover, pairwise comparisons of the two species, based on the presence or absence of unique and shared polymorphic products, were used to determine similarity coefficients, according to Jaccard (1908).
Three of partial sequences of 16S rDNA were obtained from http://blast.ncbi.nlm.nih.gov/Blast.cgi, that in additional our two sequences were used for constructing the UPGMA phylogenetic tree.Phylogenetic analyses were conducted in MEGA4 (Tamura et al., 2007).Sequence alignments were carried out using the site http://www.ebi.ac.uk/Tools/msa/clustalw2/.

For estimation of genetic diversity of two species of insects presented in the
Egyptian environment which could belong to Bracon hebetor and Bracon brevicornis we used RAPD-PCR and 16Ss rDNA sequences comparison.
RAPD-PCR is a powerful tool for the analysis of genetic diversity and was successfully used to compare genetic variation among the two species.RAPD produced by all 20 primers were used to evaluate the similarity between the studied insects as shown in Fig 1 and  Table 2.The RAPD bands were ranged from 30 to 1500 bp.The twenty primers produced total 184 bands with average 9.2 per primer.The all primers generated a total of 137 monomorphic (~74 %) and 47 polymorphic (~26%) bands in two insect under study.
The produced bands polymorphisms varied from primer to another.Among these primers, only 5 did not reveal any polymorphic bands and they produced the highly monoymorphic percentage (100%), these are identified as B18, A05, Q16, C13 and A02.On the other hand, A16, A18, A19, B15, C04 and C05 showed highest percentage of polymorphism (46%, 40%, 50%, 44%, 50% and 56%), respectively.Generally, the polymorphic percentage mean was 23.05%.Moreover, we observed variation in number of bands presented by each primer.Whereas, the primer A09 amplified the highest number of bands (15 bands) with sizes ranged from 100 to 1330 bp.While the lowest number of bands (4 bands) were produced by the A02 primer.In order to study the similarity index between insects under study, we calculated the F value of each primer.The primers B18, A05, Q16, C13 and A02 show 100% identical similarity between the two species, otherwise lowest similarities 63%, 67% and 67% were detected by using primers C05, A19 and C04, respectively.In general, mean of the similarity detected by all primers was 86%.RAPD results showed successfully the variation between the two insects.
For more clearly identification of the insects under study, we performed sequencing of mitochondrial 16S rDNA.467 bp fragment of the mitochondrial 16S rRNA gene was successfully sequenced for the two insects (Fig. 2).And the alignment of the two sequences considered in this study show several insertions or deletions and revealed many variable positions on 467 bp analyzed (Fig. 3).Moreover, the similarity between the two 16S rRNA gene sequences was 78.21%.BLAST analysis of 467 bp from the two insects which could belong to Bracon hebetor Egypt and Bracon brevicornis Egypt (presented in Egypt, figure) showed significant homology with Bracon hebetor, Callibracon limbatus and Bracon phylacteophagus.However, we did not find sequences published to Bracon brevicornis in the NCBI-Gen Bank database.The phylogenetic UPGMA tree was carried out using MEGA 4 software (Fig. 4).The UPGMA tree was constructed based on the multiply aligned sequence data for five types of insects.The tree separates the genomes into two distinct groups, whereas the insects Bracon hebetor, Callibracon limbatus, Bracon phylacteophagus and Bracon hebetor Egypt were presented in one group but only the insect Bracon brevicornis Egypt was found in another group.16S rRNA gene sequence informatics is one of the most attractive potential tools to provide genus and species identification and reclassification for the two insects under study.

DISCUSSION
The molecular studies of Bracon species have produced interesting outcomes about the hidden relationships among species which could not be observed well by the phenotypic or behavioural studies (Aruggoda, et al., 2010).RAPD-PCR and 16S rDNA gene partial region were performed in order to infer the relationship of two insects.
The RAPD-PCR results were summarized in Table 2 and they revealed genetic variations between B. hebetor Egypt and B. brevicornis Egypt.As a matter of fact, the polymorphic percentage mean and the similarity were ~23% and 86%, respectively.These results suggested that B. hebetor Egypt and B. brevicornis Egypt have a common ancestor but this percentage of polymorphism could make us to think more in their taxonomy.This difference between the two insects could due to the natural environment and it is in agreement of Jain et al. 2010 who reviewed the importance to study the insects' ecology to understand their evolution and diversification, and their influence on the functional and trophic links between different components of associated habits.Moreover, The RAPD markers technique has been reported to be an efficient tool to discriminate genetically isolated species and to verify the existence of spices that presented as a result of genetic drift or natural selection (Fuchs et al., 1998).So the RAPD marker is useful in taxonomic and classification studies (Gala, 2009).

Fig. 1 :
Fig. 1: RAPD amplified fragment produced by all 20 primers were used to evaluate the similarity and to compare genetic variation among the two insects.Heb: Bracon hebetor, B: Bracon brevicornis, M: Standard marker.

Fig. 3 :
Fig. 3: Sequences alignment of a partial mitochondrial 16S rRNA gene of the two insects by using ClustalW2 alignment.

Fig. 4 :
Fig. 4: The UPGMA tree was constructed based on the multiply aligned sequence data for two insects collected from Egyptian environment (Bracon hebetor Egypt and Bracon brevicornis Egypt) and other three insects sequences (Bracon hebetor, Callibracon limbatus and Bracon phylacteophagus published in the NCBI-GenBank database.

Table 1 :
Twenty primer sequences used in identification of two Bracon species.

Table 2 :
Polymorphisms and F value were revealed by the twenty primers that used for the identification of two Bracon species.