Molecular taxonomy

Taxonomy is the branch of science concerned with the classification of organisms. A taxonomic designation is more than just a name. Ideally, it reflects evolutionary history and the relationship between organisms. Traditionally, taxonomic classification has relied upon morphological features and physiological characteristics. However, for bacterial taxonomy, phenotypic approaches have proven insufficient. Unrelated bacteria can exhibit identical traits, closely related bacteria can have divergent features, and methods for accurate identification may be too cumbersome for routine use. In contrast, molecular taxonomy approaches use data derived from hereditary material and provide a robust view of genetic relatedness. Advances in technology have been accompanied by improvements in the cost, speed, and availability of molecular methods. Here, we provide a brief history of approaches to prokaryotic classification and describe how molecular taxonomy is redefining our understanding of bacterial evolution and the tree of life.

1. MOLECULAR TAXONOMY P MADHUSUDANA PATRA Department of zoology University of madras (Presidency college) Chennai-5

2. TAXONOMIC CHARACTERS Morphological Cytological Ethological Ecological and Biochemical

3. WHAT IS MOLECULAR TAXONOMY? The classification of organisms on the basis of the distribution and composition of chemical substances in them. Molecular (DNA,RNA, Proteins) Molecular techniques in the field of the biology have helped to establish genetic relationship between the members of different taxonomic categories.

4. MOLECULAR PHYLOGENETICS Molecular phylogenetic = the study of evolutionary relationship among biological entities (individuals, populations, species, or higher taxa)by using a combination of molecular data (such as DNA and protein sequences or absence of transposable elements, and gene-order data) and statistical techniques. Fitch and Margolish , (1967) made first phylogenetic tree based on molecular data.

5. PGYLOGENETIC TREE This tree was so close to the already established phylogenetic trees. The taxonomists realized significance of molecular data and this made them understand that other traditional methods are although important but molecular evidence could be final or confirmatory evidences. Phylogenetic studies assess the historical process which affect relationship and phylogenetic studies assess the geographical distributions. Phylogenetic and phylogeographic studies started with the introduction of Mdna markers in population genetic analysis.

6. OBJECTIVES Reconstruct the correct genealogical ties among biological entities. Estimate the time of disadvantage between biological entities. Chronicle the sequence of events along evolutionary lineage.

7. MOLECULAR MARKERS Molecular markers can be charactersized as Type I and Type II markers. Type I markers are associated with genes of known function and Type II markers are associated with genes of unknown fucntion . Allozyme markers are type I markers as the proteins they encode are associated with some functions. Microsatellites and other neutral markers are type II markers unless they are associated with genes of some known function.

8. Allozyme Allozyme electrophoresis is a method which can identify genetic variation at the level of enzymes that are directly encoded by DNA. Protein variants called allozymes originates from alleic variants and they will differ slightly in electric charge. Allozymes are codominant markers having been expressed in a heterozygous individual in a Mendelian way. Mitochondrial DNA markers Mitochondrial DNA is non-nuclear DNA in the cell having located within organelles in the cytoplasm called mitochondria. Mitochondrial DNA is maternally inherited with a haploid genome. The entire genome undergoes transcriptions as one single unit. They are not subjected to recombination and thus they are homogenous markers.

9. Microsatellites A microsatellite is a simple DNA sequence which is repeated several times across various points in the DNA OF AN ORGANISM. These repeats are highly variable and these loci can be used as markers. Single Nucleated Polymorphisms Single nucleotide polymorphism arise due to single nucleotide substitutions (transition/ transversions ) or single nucleotide insertions/deletions. These point mutations give rise to different alleles with alternative bases at a particular nucleotide position. SNPs are the most abundant polymorphisms in the genome (coding and non-coding) of any organisms. These single nucleotide variants can be detected using PCR, microchip arrays or fluorescence technology.

10. DNA microarrays or DNA chips DNA microarray consists of small glass microscope slides, silicon chip or nylon membranes with many immobilized DNA fragments arranged in a standard pattern. DNA microarrays can be utilized as a medium for matching a reporter probe of known sequence against the DNA isolated from the target sample which is of unknown origin. Species-specific DNA sequences could be incorporated to a DNA microarray and this could be used for identification purpose. DNA extracted from a target sample should be labelled with a specific fluorescent and hybridized to the microarray DNA. When the hybridization is positive a fluorescent single is detected with appropriate fluorescence scanning/imaging equipment.

11. Arbitrary Nuclear DNA markers Arbitrary markers are used when e target a segment of DNA of unknown function. The widely used methods of amplifying unknown regions are RAPD (Random Amplified P olymorphic DNA) and AFLP (Amplified Fragment Length Polymorphism) DNA. Specific Nuclear Markers Variable Numbre of Tandem Repeat is a segment if DNA that is repeated tens or even hundreds of times in nuclear genome. They repeat in tandem; vary in number in different loci and differently in individuals. There are two main classes of repetitive and highly polymorphic DNA; minisatellite DNA refebp () rring to genetic loci with repeats of length 9-56 bp and microsatellite DNA with repeats of 2-8 bp (1-6) long. Microsatellites are much more numerous in the genome of vertebrates than mini satellites.

12. Expressed Sequence Tags (ESTs) ESTs are single-pass sequences were generated from random sequencing of cDNA clones. ESTs can be used to identify genes and analyze their expression buy means of expression analysis. Fast and reliable analysis can be made for genes expression in particular tissue type under specific phenological conditions or developmental stages. Differentially expressed genes could be identified using cDNA microarrays in a systematic way. ESTs are most valuable for linkage mapping.

13. Advantage of Molecular Data Molecular entities are strictly heritable. The description of molecular characters is unambiguous. There is some regularity to the evolution of molecular traits. Molecular data are amenable to quantitative treatment. Homology assessment is easier than with morphological traits. Molecular data are robust to evolutionary distance. Molecular data are aboundant . Less time consuming.

14. DNA BARCODING PCR amplification and sequencing of a genetic marker (usually the mitochondrial COI gene).

15. ADVANTAGES Widely used in the arthropod identification Generic primers available for COI barcode is generally useful for distinguishing closely related and less closely related taxa. Alterate markers can be sequenced if COI barcode is not differential. May be useful for taxonomic analyses. DISADVANCES Require a large database of sequences for comparison Prior knowledge of the barcoding region is required when applied diagnostically Individual sequences may not provide sufficient discrimination when studying cryptic species somplexes . COI and other mitochondrial genes are maternally inherited which may result in decreased badcode diversity and skew phylogenetic inferences.

16. SPECIFIC PCR Targeted assay giving a presence or absence result for a particular genus or species. ADVANTAGES Useful diagnostically as it targets a specific taxon Can be used to target genus, species or stain within a mixed sample. No sequencing of the PCR product is required. DISADVANTAGES Require specific primer design, assay optimization and specific testing prior to use a diagnostic.

17. PCR-RFLP Involves discrimination of species based on restriction profile of amplicons.

18. ADVANTAGES Can discriminate between a range of species simultaneously. Can be used on a range of genetic markers (i.e.,; not restricted to sixe variable markers) Can provide an additional level of d iscrimination if differentiation based on size fails. May be able to detect new types in some instances DISADVANTAGES Requires downstream digestion of amplified DNA. Mutations may occasionally result in unidentified RFLP.

19. RAPD Users random primers to generate multiple PCR products resulting in a fingerprint for a particular species.

20. ADVANTAGES Simultaneously targets generic loci and is therefore more useful for discriminating closely related or cryptic species. DNA fingerprint is generated in a single reaction Data may be used for phylogenetic reconstruction in some instances. DISADVANTAGES Some issues with reproducibility. Cannot be used on mixed sample. Only useful as a diagnostic if the RAPD fingerprint of the unknown specimen has already been resolved for comparison.

21. Microsatellite Analysis Involves PCR amplification of multiple reiterated repeat-containing loci that are hypervariable due to slipped–strand mispairing mutations.

22. ADVANTAGES Simultaneously targets multiple genetic and is therefore more useful for discriminating closely related or cryptic species. When fluorescent primers are used, fragment analysis is readily automated. Assays can be multiplexed during PCR and detection (fragment analysis) phases. Some microsatellite assays can be applied across a number of different species. DISADVANTAGES Assay development is time consuming initially Cannot be used mixed samples.

23. QUANTATIVE PCR Short regions of DNA are PCR amplified and products are detected either with SYBR green (double stranded DNA dye) or via specific probes labelled with fluorescent dyes.

24. ADVANTAGES Amplification is monitored in real-time against standard of known concentration allow for quantification of target DNA. When using specific probes for amplicon detection, the reaction can be multiplexed for simoultaneous detection of up to 4-5 species and can be used in mixed samples. No electrophores is required, detection is automated and involves detection of fluorescence intensity Allow for rapid and high throughput detection. ADVANTAGES Specialized equipment required Multiplexing is limited by choice of fluorescent fyes .

25. LAMP Loop-mediated isothermal amplification. Employs 3 sets of specific primers used for amplification under isothermal conditions. Yield a ladder of amplification on electrophoresis or amplicons can be detected using SYBR green.

26. ADVANTAGES Rapid and specific amplification under isothermal conditions. Technique is potentially the most the most suitable for field conditions. Can be used with mixed samples due to primer specificity. DISADVANTAGES Assays have a relatively complex design Only suitable for field conditions when paired with a simple DNA extraction method.

27. Reference Cherul Jenkins, Toni A. Chapman, Jessica L. Micallef and Olivia L. Reynolds, 2012 molecular techniques for the detection and differentiation of Host and parasitoid species and the implications for Fruit fly Manangement . Sandhya Sukumaran and A. Gopalkrishnan , 2015 Molecular taxonomy- Application, Limitations and future. E.H Harley, Evolutionary and molecular taxonomy. Collier G. F. AND O’Brien S.J., 1985 A molecular phylogeny of the Felidae : Imminological distance, Evolution.