Hierarchical Analysis of Variation in the Mitochondrial 16SrRNA Gene among Five Different Insect Orders ()
1. Introduction
Drosophila is a model organism for research study for a longtime. However, other insects, especially pests which have a great significant value in agriculture were rarely studied. Selecting a gene for phylogenetic analysis required matching the level of sequence variation to the desired taxonomic level of study. The mitochondrial DNA of mammals had been used for molecular evolution studies [1] and similar techniques had been applied to insects as well [2] -[5] . Because of its high rate of evolution, mitochondrial DNA had been extremely useful molecule for high resolution analysis of evolutionary processes [6] . It had been used in the phylogenetic analysis of insects [7] . Factors other than the distribution of rate variation among sites could determine the shape of the sequence divergence accumulation curve. For instance, in many holometabolous insects, including Hymenoptera and Diptera, mitochondrial DNA exhibited a nucleotide composition which is strongly biased toward adenine and thymine (AT bias). For some groups, the mean percentage of AT could be higher than 80% [8] -[9] . When the base composition is biased to that degree, obviously, the ratio of transversions (tv) to transitions (ti) increased. These led to further reduce the ability to correctly estimate the number and proportion of hidden mutations and, hence, it also reduced the ability to correct sequence divergence for hidden changes. 16SrRNA of mitochondrial genome holds a crucial role in the mRNA translation and remained highly conserved throughout the evolutionary process. Here, 16SrRNA gene was selected for study across the insect orders. Primers were constructed by blasting two different genera of Diptera and were used for amplification in insect pests belonging to Diptera, Coleoptera, Heteroptera, Lepidoptera and Hymenoptera. Later, a phylogenetic tree was also constructed for understanding and analyzing the relation of five above orders.
2. Materials and Methods
2.1. Sources of Sequence Data
For 16S ribosomal partial gene sequences, I blasted Drosophila melanogaster partial 16S ribosomal RNA gene sequence (GenBank: X53506.1) and Bacterocera cucerbitae partial 16S ribosomal RNA gene sequence (GenBank: FJ168025.1). These were blasted in NCBI blast tool in Fasta format to obtain the conserved regions of 16S ribosomal RNA partial gene. Gene-specific primer used for amplification of the target partial gene was designed in-house manually. The sequence specificities of the primer sequence so designed was verified using the BLAST program available at the NCBI website (www.ncbi.nlm.nih.gov/BLAST/). The primer sequence was also evaluated for various other characteristics like melting point, presence of secondary structure formations like hairpins and propensity for dimer formation using software available in internet, Oligonucleotide Properties Calculator (http://www.basic.northwestern.edu/biotools/oligocalc.html).
2.2. Isolation of DNA
DNA was extracted from ten different insect species of five different orders (Table 1) by using Qiagen tissue kit and was verified by running in 1% agarose gel and then quantified by Spectramax M5, a dual-monochroma- tor and further processed for partial 16SrRNA gene amplification.
The PCR was carried out using Taq DNA polymerase (Fermentas) with the following general conditions: 15 - 20 Ng of genomic DNA was used in a 20 μl reaction with 5 U/μl of Taq DNA polymerase, 2.5 mm each dNTP mix and 10 pM/μl of each primer (Table 2) with the following condition.
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Table 1. Following species were considered for study.
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Table 2. Primers used for gene study.
Following an initial denaturation at 94˚C for 5 min, 37 cycles at 94˚C were performed, each annealing at 59˚C for 45 s and extension at 72˚C for 30 s. A final extension was run at 72˚C for 5 min. The PCR products (5 μl) were resolved in 1% agarose gel which was run at 80 V for 45 minutes. Negative PCR control was also run with double distilled water instead of DNA to eliminate doubts. The amplified products were eluted from the gel and purified by gel purification kit before sending for sequencing. The sequencing both through forward and reverse primers were done by Sanger method through outsourcing.
The gene sequences obtained from the samples were aligned through clustal W software in www.genome.jp/tools/clustalw/ and similarities and dissimilarities were assessed.
2.3. Construction of Phylogenetic Tree
Based on homology and genetic dissimilarities, a phylogenetic tree was constructed using rooted and branched clustal W software, www.genome.jp/tools/clustalw/.
Finally the sequences were submitted to NCBI database in www.ncbi.nlm.nih.gov/.
3. Results and Discussion
Bands amplified (Figure 1) have been verified to be ribosomal DNA by direct sequencing of the PCR products and blasting the obtained sequences with NR sequences of the NCBI database. The sequences were obtained both by forward and reverse primers and then verified and finally submitted to the NCBI database. In Coccinella septempunctata, Dysdercus koenigii, Spodoptera litura, Helicoverpa armigera and Pieris brassicae, this partial gene was reported first time. The sequences were published in NCBI database and have gene bank accession numbers (Table 3). The versatility of these ribosomal primers in insects was clearly demonstrated. Experimental studies confirmed the theoretical expectations based on sequence conservation about the relative performance of the forward and reverse primers. Since these primers amplified different orders, The identity score of all the sequences with each other were as follows: 1. Drosophila ananesse; 2. Drosophila melanogaster; 3. Drosophila jambulina; 4. Bactrocera cucurbitae; 5. Coccinella septempunctata; 6. Dysdercus koenigii; 7. Spodoptera litura; 8. Helicoverpa armigera; 9. Pieris brassicae; 10. Apis mellifera.
Sequences (1:2) Aligned. Score: 90.7834
Sequences (1:3) Aligned. Score: 95.0521
Sequences (1:4) Aligned. Score: 76.0925
Sequences (1:5) Aligned. Score: 68.0782
Sequences (1:6) Aligned. Score: 88.4106
Sequences (1:7) Aligned. Score: 67.8378
Sequences (1:8) Aligned. Score: 68.2266
Sequences (1:9) Aligned. Score: 69.4511
Sequences (1:10) Aligned. Score: 60.8466
Sequences (2:3) Aligned. Score: 86.9792
Sequences (2:4) Aligned. Score: 74.2931
Sequences (2:5) Aligned. Score: 67.7524
Sequences (2:6) Aligned. Score: 96.0265
Sequences (2:7) Aligned. Score: 64.3243
Sequences (2:8) Aligned. Score: 65.5172
Sequences (2:9) Aligned. Score: 68.9737
Sequences (2:10) Aligned. Score: 60.0529
Sequences (3:4) Aligned. Score: 70.0521
Sequences (3:5) Aligned. Score: 52.1173
Sequences (3:6) Aligned. Score: 86.4238
Sequences (3:7) Aligned. Score: 57.2973
Sequences (3:8) Aligned. Score: 66.6667
Sequences (3: 9) Aligned. Score: 60.9375
Sequences (3:10) Aligned. Score: 56.8783
Sequences (4:5) Aligned. Score: 58.9577
Sequences (4:6) Aligned. Score: 75.4967
Sequences (4:7) Aligned. Score: 58.9189
Sequences (4:8) Aligned. Score: 56.0411
Sequences (4:9) Aligned. Score: 60.1542
Sequences (4:10) Aligned. Score: 47.8836
Sequences (5:6) Aligned. Score: 55.6291
Sequences (5:7) Aligned. Score: 65.798
Sequences (5:8) Aligned. Score: 56.6775
Sequences (5:9) Aligned. Score: 71.6612
Sequences (5:10) Aligned. Score: 50.8143
Sequences (6:7) Aligned. Score: 60.596
Sequences (6:8) Aligned. Score: 65.894
Sequences (6:9) Aligned. Score: 68.2119
Sequences (6:10) Aligned. Score: 54.3046
Sequences (7:8) Aligned. Score: 70.2703
Sequences (7:9) Aligned. Score: 77.5676
Sequences (7:10) Aligned. Score: 52.7027
Sequences (8:9) Aligned. Score: 65.5172
Sequences (8:10) Aligned. Score: 61.9048
Sequences (9:10) Aligned. Score: 60.582
As per the above score and the phylogenetic tree (Figure 2), it was clear that though, Drosophila ananesse, Drosophila melanogaster, Drosophila jambulina and Bactrocera cucurbitae belonged to same order Diptera, all the species of Drosophila in terms of partial 16SrRNA gene sequence were evolutionary more close to Dysdercus
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Figure 1. Distinct amplification of samples approximately corresponds to the 500 bp of the DNA marker (extreme right).
koenigii of order Heteroptera than Bactrocera cucurbitae. Bactrocera cucurbitae was placed in different branch and Dysdercus koenigii was placed in same branch of phylogenetic tree with respect to all species of Drosophila. Similarly, Spodoptera litura, Helicoverpa armigera and Pieris brassicae belonged to same order Lepidoptera. Spodoptera litura and Helicoverpa armigera belonged to same family Noctuidae whereas Pieris brassicae belonged to family Pieridae. The results showed that Spodoptera litura in terms of partial 16SrRNA gene sequence was evolutionary more close to Pieris brassicae. Spodoptera litura and Pieris brassicae were placed in same branch whereas Helicoverpa armigera was placed in different branch of the phylogenetic tree. Although we could not judge the sequence similarities by studying a partial sequence of 16SrRNA, this finding was unexpected as it was expected that genera of same orders and families were evolutionary close to each other. Similar results were showed by Shouche et al. [10] , where mitochondrial 16SrRNA gene fragment was analyzed in mosquito. A phylogenetic tree was displayed and it showed that all studied species of Aedes, Culex and Anopheles belonging to same order Diptera and same family Culicidae were not in the same branch and thus might have had different evolutionary origins.
4. Conclusion
Insects having same taxonomic group might have different evolutionary origin in respect of a partial conserved gene, 16SrRNA.
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Table 3. Name of the organism with accession numbers.
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Figure 2. Phylogenetic tree of the above sequences based on rooted and branched clustal W software.
Acknowledgements
The corresponding author is thankful to DST for funding (DST Fast Track Project).