A Contribution of ICAR National Fellow Project

दीमक से बचाव के लिए देशी तकनीकPopularising the Termitexpert web-portal

Molecular Taxonomy

Termite mitochondrial genes – isolation and characterization

DNA was extracted from termite specimens using Nucleospin tissue DNA Extraction kit. Four markers (12S rRNA, 16S rRNA, cytochrome b, cytochrome oxidase c- subunit-II) were amplified from mitochondrial and nuclear DNA. PCR amplifications performed, products fractionated on agarose gel, verified for specificity of the band, and purified using promega gel extraction kit; sequenced and subsequently submitted to GenBank (NCBI). Acquired accession numbers from NCBI are listed (total of 22 accessions).

Molecular Characterization of Termites

Traditional identification of termite is difficult and sometimes unreliable due to the morphological ambiguity among the termite species. Mitochondrial genes are known as conserved one and their DNA is more abundantly available compare to nuclear genes. Polymerase chain reaction (PCR)-based techniques readily separate closely related taxa by their taxon-specific mitochondrial DNA (mtDNA) markers. The Universal primers rRNA (eg. 12S and 16S) and protein coding genes (e.g., cytochrome oxidase CO I and II) are the primary mtDNA markers used in molecular identification. The Mitochondrial 12S rDNA, 16S rDNA, 28S rRNA, 5.8S rRNA, Cytochrome oxidase subunit I (Barcode), Cytochrome oxidase subunit II, Cytochrome b and NADH dehydrogenase subunit 1 (NAD1) genes are the efficient biomarker to identify and explore the phylogeny of termite. For instance, recent study proves that the nucleotide sequences in a small area of the cytochrome oxidase unit I (COI) is highly conservative and differs among most of the species and has been sequenced in many different groups because it contains highly conserved functional domain and its rate of nucleotide evolution allows it to resolve evolutionary histories at the family, genus, and species levels.

It is hearty to cite that nuclear and mitochondrial genes of some major Indian termites have already been sequenced and accession numbers are available in NCBI. The work done is by Sobti et al. (2009, Molecular & Cellular Biochemistry 331: 145-151), Singla et al. (2013, International Journal of Evolution 2:1) during 2007-2012 (http://www.ncbi.nlm.nih.gov/). Recently Srinivas et al. (2015, Journal of Zoological Research 4(1):1-6) investigated on the genetic relationship among termites collected from various locations of India based on 12S rRNA gene.  We acquired thirteen accessions for different species of higher termites in 2013-15. We sequenced 12S rRNA, 16S rRNA and Cytochrome oxidase subunit II (COII) gene of these termites whose accession numbers are highlighted in the table below. We optimized PCR conditions for all three genes and the PCR programming is mentioned in table.  The PCR reactions were set up in a 50 μL reaction mixture containing 10 μL of lOX PCR buffer, 1.5 μL of 2.5 mM dNTPs, 1 μL of forward PCR primer (20 ng/μL), 1 μL of reverse PCR primer (20 ng/μL), 1.0 μL Taq Polymerase, and 4 μL template DNA and make upto 50 μl by adding sterile distilled water. PCR product was sequenced and the resulting trimmed nucleotide sequence was submitted in NCBI. We strive for the same work for rest of the Indian termite species those were not sequenced.

PCR programming used for amplification of respective gene

We present here the comprehensive table for the molecular characterization of some Indian termites. The table was prepared by searching all gene sequences of termites in NCBI only for India.  Analysis of all sequences was done by doing complete alignment in Mega 4.1 (Beta 3) software, in order to obtain a lucid representation giving complete information of all the submitted termite sequences from India. We manually and meticulously searched accessions of all genes for Indian termite species reported till date. It was found that 101 gene sequences of termites were submitted by Indian researchers in NCBI till-date (Sept 2015), out of which, 38 sequences were of 12S rRNA gene, 24 sequences were of 16S rRNA gene, 11 sequences were of 28S rRNA gene, 13 sequences were of NADH dehydrogenase (ND1) gene, 3 sequences were of 5.8S rRNA gene, 7 sequences were of COII gene and only 5 sequence were of COI gene. Since, Cytochrome oxidase subunit I (COI) marker is difficult to amplify due to large size (approx. 1179bp), only 5 sequences were submitted in NCBI till date.

Out of these 101 gene sequences, 22 are from our laboratory (NF Project work exclusively).

Gene Sequences acquired from NCBI:

KT820661, KP765715, KP748241, KT820658, KP410731, KP410732, KP864045, KP864044, KR296660, KT820659, KF170428, KT820657, KR078331, KT828709, KT820659, KT828710, KT828711, KP780274, KR078330, KF170427, HF968496, KF445388

Last Updated: 04-01-2020

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