**4. Discussion**

122 Biodiversity Conservation and Utilization in a Diverse World

**Figure 10.** Multiple sequence alignments result showing the total variable sites between river buffalo (BBU), Cattle (Bost, Bosi and JBC), Sheep (Ovis) and goat (Capra) in the specific 16S rRNA fragment. Sequences of the 16S rRNA fragment of Egyptian buffaloes (FJ748599–FJ748607). Differing nucleotides

are noted (T, A, G and C)

#### **4.1. DNA barcoding, genome evolution & phylogenetic trees**

The ability of molecular trees to encompass both short and long periods of time is based on the observation that different genes evolve at different rates. The DNA specifying ribosomal RNA (rRNA) changes relatively slowly, so comparisons of DNA sequences in these genes are useful for investigating relationships between taxa that diverged hundreds of millions of years ago. Studies of the genes for rRNA have shown, for example, that fungi are more closely related to animals than to green plants—something that certainly could not have been deduced from morphological comparisons alone.

In contrast, the DNA in mitochondria (mtDNA) evolves relatively rapidly and can be used to investigate more recent evolutionary events.

The methodology used in DNA barcoding has been straightforward. Sequences of the barcoding region are obtained from various individuals. The resulting sequence data are then used to construct a phylogenetic tree using a distance-based 'neighbour-joining' method. In such a tree, similar, putatively related individuals are clustered together. The term 'DNA barcode' seems to imply that each species is characterized by a unique sequence,

but there is of course considerable genetic variation within each species as well as between species. However, genetic distances between species are usually greater than those within species, so the phylogenetic tree is characterized by clusters of closely related individuals, and each cluster is assumed to represent a separate species.

Biological Identifications Through DNA Barcodes 125

amplification. A short DNA sequence of 600 bp in the mitochondrial gene for cytochrome *c*  oxidase subunit 1 (CO1) has been accepted as a practical, standardized species-level barcode for animals (see www.barcoding.si.edu). The inability of CO1 to work as a barcode in plants set off a race among botanists to find a more appropriate marker. A number of candidate gene regions have been suggested as possible barcodes for plants, but none have been widely accepted by the taxonomic community. This lack of consensus is in part due to the limitations inherent in a plastid marker relative to plant CO1, and also because a quantitative context for selecting a gene region as a barcode for plants has not been offered. Several factors must be considered and weighted in selecting a plant DNA barcode: (*i*) universal PCR amplification, (*ii*) range of taxonomic diversity, (*iii*) power of species

Evolution results from the accumulation of inherited changes in populations. Because DNA is the molecule of heredity, evolutionary changes must be reflected in changes in DNA. Systematics have long known that comparing DNA within a group of species would be a powerful method for inferring evolutionary relationships, but for most of the history of systematics, direct access to genetic information was nothing more than a dream. Today, however, **DNA sequencing**—determining the sequence of nucleotides in segment of DNA – is comparatively cheap, easy, and widely available. *The polymerase chain reaction* (PCR) allows systematics to easily accumulate large samples of DNA from organisms, and

automated machinery makes sequence determination a comparatively simple task.

i. make the outputs of systematics available to the largest possible community of endusers by providing standardized and high-tech identification tools, e.g. for biomedicine (parasites and vectors), agriculture (pests), environmental assays and customs (trade in

ii. relieve the enormous burden of identifications from taxonomists, so they can focus on more pertinent duties such as delimiting taxa, resolving their relationships and

Perhaps another advantage of DNA barcoding is that it will also facilitate basic biodiversity inventories. Indeed, from the premises of molecular phylogenetics to assembling the tree of life, DNA sequences in environmental sampling and reconstruction of phylogenetic trees to place sequences into an evolutionary context have been used in several inventories of

New 'Genetic Bar Code' Technique Establishes Ability to Derive DNA Information from

**4.3. Direct benefits of DNA barcoding undoubtedly include** 

iii. pair up various life stages of the same species (e.g. seedlings, larvae);

cryptic biodiversity (e.g. soil bacteria or marine/freshwater micro-organisms).

endangered species);

RNA

discovering and describing new species;

iv. provide a bio-literacy tool for the general public.

differentiation, and (*iv*) bioinformatics analysis and application.

**4.2. Molecular genetics reveals evolutionary relationships** 

An evolutionary tree (or *phylogenetic tree*) is a branching diagram that represents the evolutionary history of a group of organisms. For example, we might use morphological and genetic data to figure out a phylogenetic tree of animals. Such a tree can provide a huge amount of information. For any particular group of animals our tree could identify the ancestors and closest relatives of the group. If we traced the history of animals all the way back, we could use the tree to help us answer questions such as, What did the earliest animals look like? What features did they pass on to all their descendants?. Phylogenetic trees also have great practical value. The same techniques we use to reconstruct evolutionary history have been used in forensics, where phylogenetic trees have helped solve criminal cases, and epidemiology, where trees have been used to estimate when and where diseases such as AIDS originated.

Now that we can compare entire genomes, including our own, some interesting facts have emerged. As you may have heard, the genomes of humans and chimpanzees are strikingly similar. An even more remarkable fact is that homologous genes are widespread and can extend over huge evolutionary distances. While the genes of humans and mice are certainly not identical, 99% of them are detectably homologous. And 50% of human genes are homologous with those of yeast.

It is not a coincidence that DNA barcoding has developed in concert with genomics-based investigations.

DNA barcoding (a tool for rapid species identification based on DNA sequences) and genomics (which compares entire genome structure and expression) share an emphasis on large scale genetic data acquisition that offers new answers to questions previously beyond the reach of traditional disciplines. DNA barcodes consist of a standardized short sequence of DNA (400–800 bp) that in principle should be easily generated and characterized for all species on the planet (1). A massive on-line digital library of barcodes will serve as a standard to which the DNA barcode sequence of an unidentified sample from the forest, garden, or market can be matched. Similar to genomics, which has accelerated the process of recognizing novel genes and comparing gene function, DNA barcoding will allow users to efficiently recognize known species and speed the discovery of species yet to be found in nature. DNA barcoding aims to use the information of one or a few gene regions to identify all species of life, whereas genomics, the inverse of barcoding, describes in one (e.g., humans) or a few selected species the function and interactions across all genes.

To be practical as a DNA barcode a gene region must satisfy three criteria: (*i*) contain significant species-level genetic variability and divergence, (*ii*) possess conserved flanking sites for developing universal PCR primers for wide taxonomic application, and (*iii*) have a short sequence length so as to facilitate current capabilities of DNA extraction and amplification. A short DNA sequence of 600 bp in the mitochondrial gene for cytochrome *c*  oxidase subunit 1 (CO1) has been accepted as a practical, standardized species-level barcode for animals (see www.barcoding.si.edu). The inability of CO1 to work as a barcode in plants set off a race among botanists to find a more appropriate marker. A number of candidate gene regions have been suggested as possible barcodes for plants, but none have been widely accepted by the taxonomic community. This lack of consensus is in part due to the limitations inherent in a plastid marker relative to plant CO1, and also because a quantitative context for selecting a gene region as a barcode for plants has not been offered. Several factors must be considered and weighted in selecting a plant DNA barcode: (*i*) universal PCR amplification, (*ii*) range of taxonomic diversity, (*iii*) power of species differentiation, and (*iv*) bioinformatics analysis and application.

#### **4.2. Molecular genetics reveals evolutionary relationships**

124 Biodiversity Conservation and Utilization in a Diverse World

where diseases such as AIDS originated.

homologous with those of yeast.

investigations.

and each cluster is assumed to represent a separate species.

but there is of course considerable genetic variation within each species as well as between species. However, genetic distances between species are usually greater than those within species, so the phylogenetic tree is characterized by clusters of closely related individuals,

An evolutionary tree (or *phylogenetic tree*) is a branching diagram that represents the evolutionary history of a group of organisms. For example, we might use morphological and genetic data to figure out a phylogenetic tree of animals. Such a tree can provide a huge amount of information. For any particular group of animals our tree could identify the ancestors and closest relatives of the group. If we traced the history of animals all the way back, we could use the tree to help us answer questions such as, What did the earliest animals look like? What features did they pass on to all their descendants?. Phylogenetic trees also have great practical value. The same techniques we use to reconstruct evolutionary history have been used in forensics, where phylogenetic trees have helped solve criminal cases, and epidemiology, where trees have been used to estimate when and

Now that we can compare entire genomes, including our own, some interesting facts have emerged. As you may have heard, the genomes of humans and chimpanzees are strikingly similar. An even more remarkable fact is that homologous genes are widespread and can extend over huge evolutionary distances. While the genes of humans and mice are certainly not identical, 99% of them are detectably homologous. And 50% of human genes are

It is not a coincidence that DNA barcoding has developed in concert with genomics-based

DNA barcoding (a tool for rapid species identification based on DNA sequences) and genomics (which compares entire genome structure and expression) share an emphasis on large scale genetic data acquisition that offers new answers to questions previously beyond the reach of traditional disciplines. DNA barcodes consist of a standardized short sequence of DNA (400–800 bp) that in principle should be easily generated and characterized for all species on the planet (1). A massive on-line digital library of barcodes will serve as a standard to which the DNA barcode sequence of an unidentified sample from the forest, garden, or market can be matched. Similar to genomics, which has accelerated the process of recognizing novel genes and comparing gene function, DNA barcoding will allow users to efficiently recognize known species and speed the discovery of species yet to be found in nature. DNA barcoding aims to use the information of one or a few gene regions to identify all species of life, whereas genomics, the inverse of barcoding, describes in one (e.g.,

humans) or a few selected species the function and interactions across all genes.

To be practical as a DNA barcode a gene region must satisfy three criteria: (*i*) contain significant species-level genetic variability and divergence, (*ii*) possess conserved flanking sites for developing universal PCR primers for wide taxonomic application, and (*iii*) have a short sequence length so as to facilitate current capabilities of DNA extraction and Evolution results from the accumulation of inherited changes in populations. Because DNA is the molecule of heredity, evolutionary changes must be reflected in changes in DNA. Systematics have long known that comparing DNA within a group of species would be a powerful method for inferring evolutionary relationships, but for most of the history of systematics, direct access to genetic information was nothing more than a dream. Today, however, **DNA sequencing**—determining the sequence of nucleotides in segment of DNA – is comparatively cheap, easy, and widely available. *The polymerase chain reaction* (PCR) allows systematics to easily accumulate large samples of DNA from organisms, and automated machinery makes sequence determination a comparatively simple task.

#### **4.3. Direct benefits of DNA barcoding undoubtedly include**


Perhaps another advantage of DNA barcoding is that it will also facilitate basic biodiversity inventories. Indeed, from the premises of molecular phylogenetics to assembling the tree of life, DNA sequences in environmental sampling and reconstruction of phylogenetic trees to place sequences into an evolutionary context have been used in several inventories of cryptic biodiversity (e.g. soil bacteria or marine/freshwater micro-organisms).

New 'Genetic Bar Code' Technique Establishes Ability to Derive DNA Information from RNA

*Science Daily* (Apr. 8, 2012) — Researchers from Mount Sinai School of Medicine have developed a method to derive enough DNA information from non-DNA sources -- such as RNA -- to clearly identify individuals whose biological data are stored in massive research repositories. The approach may raise questions regarding the ability to protect individual identity when high-dimensional data are collected for research purposes.

Biological Identifications Through DNA Barcodes 127

[1] Pascal G, Mahe S (2001) Identity, traceability, acceptability and substantial equivalence

[2] Skarpeid HJ, Kvaal K, Hildrum KI (1998) Identification of animal species in ground meat mixtures by multivariate analysis of isoelectric focusing protein profiles.

[3] 3. Hsieh YH, Sheu SC, Bridgman RC (1998) Development of a monoclonal antibody

[4] Ashmoor SH, Monte WC, Stiles PG (1998) Liquid chromatographic identification of

[5] Parson W, Pegoraro K, Niederstatter H, Fo¨ger M, Steinlechner M (2000) Species

[6] Hsieh HM, Chiang HL, Tsai LC, Lai SY, Huang NE, Linacre A, Lee JC (2001) Cytochrome b gene for species identification of the conservation animals. Forensic Sci

[7] Murray BW, McClymont RA, Strobeck C (1995) Forensic identification of ungulate species using restriction digests of PCRamplified mitochondrial DNA. J Forensic Sci

[8] Balitzki-Korte B, Anslinger K, Bartsch C, Rolf B (2005) Species identification by means of pyrosequencing the mitochondrial 12S rRNA gene. Int J Legal Med 119:291–294 [9] Rodr´guez MA, Garc´a T, Gonza´lez I, Asensio L, Herna´ndez PE, Mart´n R (2004) PCR identification of beef, sheep, goat, and pork in raw and heat-treated meat mixtures.

[10] Rodr´guez MA, Garc´a T, Gonza´lez I et al (2003) Identification of goose, mule, duck, chicken, turkey, and swine in foie gras by species-specific polymerase chain reaction. J

[11] Montiel-Sosa JF, Ruiz-Pesini E, Montoya J, Roncale´s P, Lo´pez- Pe´rez MJ, Pe´rez-Martos A (2000) Direct and highly speciesspecific detection of pork meat and fat in meat products by PCR amplification of mitochondrial DNA. J Agric Food Chem

[12] Ramadan HAI, El Hefnawi M (2008) Phylogenetic analysis and comparison between cow and buffalo (including Egyptian buffaloes) mitochondrial displacement-loop

[13] Waugh J (2007) DNA barcoding in animal species: progress, potential and pitfalls.

[14] Hebert PDN, Cywinska A, Ball SL, de Waard JR (2003) Biological identifications

[15] Hebert PDN, Stoeckle MY, Zemlak TS, Francis CM (2004) Identification of birds

[16] Ward RD, Zemlak TS, Innes BH, Last PR, Hebert PDN (2005) DNA barcoding

Australia's fish species. Philos Trans R Soc Lond B 360:1847–1857

identification by means of the cytochrome b gene. Int J Legal Med 114:23–28

specific to cooked mammalian meats. J Food Prot 61(4):476–487

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regions. Mitochondrial DNA 19(4):401–410

through DNA barcodes. PLoS Biol 2:E312

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of food. Cell Mol Biol 47:1329–1342

meats. J Assoc Off Anal Chem 71:397–403

Electrophoresis 19:3103–3109

A paper introducing the technique appears in the April 8 online edition of *Nature Genetics*.

DNA contains the genetic instructions used in the development and functioning of every living cell. RNA acts as a messenger that relays genetic information in the cell so that the great majority of processes needed for tissue to function properly can be carried out.

To date, access to databases with DNA information has been restricted and protected as it has long been considered the sole genetic fingerprint for every individual. However, vast amounts of RNA data have been made publicly available via a number of databases in the United States and Europe. These databases contain thousands of genomic studies from around the world.

In this study, authors developed a technique whereby a person's DNA could be inferred from RNA data using gene-expression levels monitored in any of a number of tissues. In contrast, most studies involving DNA and RNA begin with DNA sequences and then seek to associate expression patterns with changes in DNA between individuals in a population. This is the first time going from RNA levels to DNA sequence has been described.

"By observing RNA levels in a given tissue, we can infer a genotypic barcode that uniquely tags an individual in ways that enables matching the individual to an independently derived DNA sample,". Not only can genotypic barcodes be deduced from RNA, but RNA levels in some tissue can inform not only individual characteristics like age and sex, but on diseases such as Alzheimer's and cancer, as well as the risks of developing those diseases."
