The North East India is a reservoir of rich biodiversity for much of India’s flora and fauna, and as a consequence, the region is one of the richest in biological values. DNA based species identification or DNA barcoding is an exciting tool for documenting biodiversity with a gene sequence. Here we focused on the development of DNA barcode sequence of Greater Indian one-horned Rhinoceros [Rhinoceros unicornis] by determining the DNA sequence of its mitochondrial 5´ COI gene region using cross primer design for PCR amplification. The cross species primer pairs of forward primer fish [FISH COX F] and reverse primer invertebrate [INV R] DNA barcoding, successfully amplified the barcode sequence of R. unicornis which is confirmed through similarity search in National Centre for Biotechnology [NCBI, www.ncbi.nlm.nih.gov] and Barcode of life [BOLD] database and submitted to NCBI-GenBank with accession no. JN417004 is the first COI barcode sequence of R. unicornis of Greater Indian one-horned Rhino. We observed the maximum identification of the query sequence in case of R. unicornis (99%) and R. sondaicus (93%). Mean pair wise distance was computed using K2P to check the inter species divergence between all species in each genus among the members of Rhinoceretidae family in COI region taking 632 bp into consideration, within species was found to be 0.002 as the maximum and 0.000 as the minimum and that of in between species was found to be 0.064 as the maximum and 0.000 as the minimum. The study represents development of COI species specific DNA barcode of R. unicornis with regard to conservation and trade aspect. The cross species primer pair to amplify barcode gene for a species for which no specific primer are available represents a noble approach for barcoding on those areas like North East India where biological diversity is high.
Table of Contents
CHAPTER - 1 Introduction
CHAPTER - 2 Review of literature
CHAPTER - 3 Materials and methods
CHAPTER - 4 Results
CHAPTER - 5 Discussion
Research Objectives and Core Themes
The primary objective of this research is to develop a species-specific DNA barcode sequence for the Greater Indian One-Horned Rhinoceros (Rhinoceros unicornis) to assist in conservation efforts through reliable molecular species identification. The study focuses on optimizing cross-species primer design for mitochondrial COI gene amplification and conducting phylogenetic analysis among the Rhinoceretidae family to confirm taxonomic identity.
- Development of cross-species PCR primers for mitochondrial COI amplification.
- Isolation and sequencing of the COI barcode region for Rhinoceros unicornis.
- Bioinformatics analysis of nucleotide composition and phylogenetic relationships.
- Application of DNA barcoding for wildlife forensics and conservation.
Excerpt from the Book
INTRODUCTION
The biological diversity of each country is a valuable and vulnerable natural resource. The North East of India comprising of the 8 sister states of Assam, Arunachal Pradesh, Manipur, Meghalaya, Mizoram, Nagaland, Tripura and Sikkim, is a reservoir of rich biodiversity for much of India’s flora and fauna, and as a consequence, the region is one of the richest in biological values. But North East India has still been able to retain it’s a significant proportion of its biodiversity, possibly due to long years of isolation and difficult terrain but is now under increasing pressure to restrain its resources.
DNA based species identification or in other words DNA barcoding is an exciting tool for documenting biodiversity with a gene sequence. Here we focus our interest on the development of DNA barcode sequence of Greater Indian Rhinoceros (Rhinoceros unicornis) as this species is now restricted to Kaziranga, Pabitora and Orang in Assam of Northeast India. The rhinoceros is a large, primitive looking mammal that in fact dates from the Miocene era millions of years ago and is characterized by a snout with one or two horns belong to the order Perissodactyla & family Rhinoceretidae.
Summary of Chapters
CHAPTER - 1 Introduction: This chapter establishes the importance of biodiversity in Northeast India and introduces DNA barcoding as a critical tool for species identification and conservation of the endangered Indian Rhinoceros.
CHAPTER - 2 Review of literature: The section provides an overview of existing research on DNA barcoding, the functionality of the COI gene in species identification, and the role of bioinformatics platforms like BOLD and NCBI in taxonomic analysis.
CHAPTER - 3 Materials and methods: This chapter details the laboratory procedures, including genomic DNA isolation from hair follicles, cross-species primer selection, PCR optimization, and the specific software tools used for sequence analysis.
CHAPTER - 4 Results: This chapter presents the experimental data, including the successful amplification of the COI barcode, the resulting nucleotide sequence for Rhinoceros unicornis, and findings from phylogenetic and genome analysis.
CHAPTER - 5 Discussion: The final chapter evaluates the success of the study in developing the first COI barcode for Rhinoceros unicornis and discusses the broader implications for wildlife forensics and conservation management.
Keywords
DNA Barcoding, Rhinoceros unicornis, Cytochrome Oxidase Subunit I, COI, Conservation, PCR, Phylogenetic Analysis, Rhinoceretidae, Mitochondrial DNA, Bioinformatics, Species Identification, Molecular Markers, Northeast India, Genetic Divergence.
Frequently Asked Questions
What is the core focus of this research project?
The project focuses on developing a standardized DNA barcode sequence for the Greater Indian One-Horned Rhinoceros to facilitate accurate species identification, which is critical for legal protection and conservation.
What are the central thematic areas of the study?
The study centers on mitochondrial DNA analysis, molecular identification techniques, cross-species primer design, and the use of bioinformatics tools for phylogenetic reconstruction.
What is the primary goal of the research?
The primary goal is the successful sequencing of the COI barcode region of Rhinoceros unicornis and the validation of its taxonomic identity within the Rhinoceretidae family.
Which scientific methods were employed?
The researchers used conventional phenol-chloroform DNA isolation, PCR optimization with custom-designed cross-species primers, and advanced bioinformatics platforms including BLAST, MEGA5, and BOLD.
What does the main body of the work cover?
The main body covers the theoretical background of DNA barcoding, detailed laboratory protocols for molecular analysis, the presentation of generated sequence data, and comparative phylogenetic studies.
Which keywords best characterize this work?
The work is characterized by terms such as DNA Barcoding, Rhinoceros unicornis, COI, Bioinformatics, Phylogenetic Analysis, and Conservation.
Why was the COI gene chosen for this study?
The COI gene was selected because of its proven effectiveness as a universal molecular marker for animal species identification, its ease of amplification across different taxa, and its significant sequence variation.
What is the significance of the accession number JN417004?
This is the official GenBank accession number for the first COI barcode sequence of the Greater Indian One-Horned Rhinoceros, which was generated and submitted as a result of this study.
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- Research Scholar Tarikul Mazumder (Autor), 2014, Identification of Species Specific DNA Marker as Barcode Sequence from Greater Indian One-Horned Rhinoceros (Rhinoceros unicornis) from North East India, Múnich, GRIN Verlag, https://www.grin.com/document/278349
