MOLECULAR TAXONOMY

Submitted by-M.IllakeyaI M.Sc PHE

Molecular Taxonomy

Taxonomic Characters

Morphological, Cytological, Ethological, Ecological and Biochemical characters.

Molecular Taxonomy

What Is Molecular Taxonomy?

The classification of organisms on the basis of the distribution and composition of chemical substances in them.

Molecular techniques in the field of biology have helped to establish genetic relationship between the members of different taxonomic categories.

Molecular Taxonomy

Molecular (DNA, RNA, proteins)

Molecular phylogenetics Molecular phylogenetic = The study of evolutionary relationships among biological entities (individuals, populations, species, or higher taxa), by using a combination of molecular data (such as DNA and protein sequences, presence or absence of transposable elements, and gene-order data) and statistical techniques.

Fitch and Margoliash ,(1967) made first phylogenetic tree based on molecular data.

Molecular Taxonomy

Phylogenetic 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 evidences could be final or confirmatory evidences.

Phylogenetic studies assess the historical processes which affect relationships and phylogeographic studies assess the geographical distributions.

Phylogenetic and phylogeographic studies started with the introduction of mtDNA markers in population genetic analyses.

Molecular Taxonomy

Objectives Reconstruct the correct genealogical ties

among biological entities

Estimate the time of divergence between biological entities

Chronicle the sequence of events along evolutionary lineages

Molecular Taxonomy

Molecular markers

Molecular markers can be characterized as Type I and Type II markers;

1) Type I markers are associated with genes of known function and

2) Type II markers are associated with genes of unknown function.

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.

Molecular Taxonomy

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 allelic 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

in within organelles in the cytoplasm called mitochondria. Mitochondrial DNA is maternally inherited with a haploid genome.

The entire genome undergoes transcription as one single unit. They are not subjected to recombination and thus they are homologous markers.

Molecular Taxonomy

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 Nucleotide Polymorphisms Single nucleotide polymorphisms arise due to single

nucleotide substitutions (transitions/transversions) or single nucleotide insertions/deletions.

These point mutations give rise to different alleles with alternative bases at a particular nucleotide position.

SNP,s are the most abundant polymorphisms in the genome (coding and non-coding) of any organism.

These single nucleotide variants can be detected using PCR, microchip arrays or fluorescence technology

Molecular Taxonomy

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.

A DNA microarray 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 purposes.

DNA extracted from a target sample should be labeled with a specific fluorescent molecule and hybridized to the microarray DNA.

When the hybridization is positive a fluorescent signal is detected with appropriate fluorescence scanning/imaging equipment. Molecular Taxonomy

Arbitrary Nuclear DNA markers Arbitrary markers are used when we target a segment of DNA of

unknown function. The widely used methods of amplifying unknown regions are RAPD

(Random Amplified Polymorphic DNA) and AFLP (Amplified Fragment Length Polymorphism) DNA.

Specific Nuclear DNA markers Variable Number of Tandem Repeat is a segment of DNA that is

repeated tens or even hundreds to thousands 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 referring to genetic loci with repeats of length 9-65 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.

Molecular Taxonomy

Expressed Sequence Tags (ESTs)

ESTs are single-pass sequences which were generated from random sequencing of cDNA clones.

ESTs can be used to identify genes and analyze their expression by means of expression analysis.

Fast and reliable analysis can be made for the genes expressed in particular tissue types under specific physiological conditions or developmental stages.

Differentially expressed genes could be identified using cDNA microarrays in a systematic way.

ESTs are most valuable for linkage mapping.

Molecular Taxonomy

Advantages 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 abundant. Less time consuming

Molecular Taxonomy

DNA barcoding PCR amplification and sequencing of a

genetic marker (usually the mitochondrial COI gene)

Molecular Taxonomy

Advantages Widely used in arthropod identification Generic primers available for COI barcode region COI is

generally useful for distinguishing closely related and less closely related taxa

Alternate markers can be sequenced if COI barcode is not differential

May be useful for taxonomic analysesDisadvantages Requires 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 complexes COI and other mitochondrial genes are maternally inherited which

may result in decreased barcode diversity and skew phylogenetic inferences

Molecular Taxonomy

Specific PCR Targeted assay giving a presence or absence result for

a particular genus or speciesAdvantages Useful diagnostically as it targets a specific taxon Can be used to target a specific genus, species or

strain within a mixed sample No sequencing of the PCR product is requiredDisadvantages Requires specific primer design, assay optimization

and specificity testing prior to use as a diagnostic

Molecular Taxonomy

Size differential PCR Employs generic PCR primers but yields

amplicons that differ in length. Usually targets the intergenic transcribed spacer regions (ITS).

Molecular Taxonomy

Advantages Can discriminate between a range of species

simultaneously Differentiation is by electrophoresis, Does not required sequencing of the amplicon Disadvantages Size of amplicon needs to vary substantially to

enable discrimination ITS regions contain repetitive regions that can

result in PCR products with multiple bands

Molecular Taxonomy

PCR-RFLP Involves discrimination of species based

on restriction profile of amplicons.

Molecular Taxonomy

Advantages Can discriminate between a range of species

simultaneously Can be used on a range of genetic markers (i.e.,: not

restricted to size variable markers) Can provide an additional level of discrimination if

differentiation based on size fails May be able to detect new types in some instancesDisadvantages Requires downstream digestion of amplified DNA Mutations may occasionally result in unidentified

RFLP patterns

Molecular Taxonomy

Multiplex PCR Combines multiple primer sets with

different specificities in a single assay

Molecular Taxonomy

Advantages Detects and differentiates multiple species in a single

assay Can be used on multiple genetic markers Discrimination is by electrophoretic size differentiation,

so no downstream processing of amplicons is required Useful for simultaneous detection of species in mixed

samples (e.g., detection of host and parasitoid DNA in one assay).

Disadvantages Can be difficult assays to optimize due to the presence

of multiple primer sets Potential cross-hybridization of primers may interfere

with reaction

Molecular Taxonomy

RAPD Uses random primers to generate multiple

PCR products resulting in a fingerprint for a particular species.

Molecular Taxonomy

Advantages Simultaneously targets multiple genetic 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 instancesDisadvantages Some issues with reproducibility Cannot be used on mixed samples. Only useful as a diagnostic if the RAPD fingerprint of

the unknown specimen has already been resolved for comparison

Molecular Taxonomy

AFLP Involves ligation of adaptors to digested

DNA followed by PCR amplification using primers that are partially adaptor and partially gene-specific

Molecular Taxonomy

Advantages Simultaneously targets multiple genetic loci and is

therefore more useful for discriminating closely related or cryptic species

Very sensitive and more robust than RAPD Data may be used for phylogenetic reconstruction in

some instancesDisadvantages Requires manipulations in addition to PCR Cannot be used on mixed samples. Only useful as a diagnostic if the AFLP fingerprint of

the unknown specimen has already been resolved for comparison

Molecular Taxonomy

Microsatellite analysis Involves PCR amplification of multiple

reiterated repeat-containing loci that are hypervariable due to slipped-strand mispairing mutations

Molecular Taxonomy

Molecular Taxonomy

Advantages Simultaneously targets multiple genetic loci 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 on mixed samples.

Molecular Taxonomy

Quantitative PCR Short regions of DNA are PCR amplified

and products are detected either with SYBR green (double stranded DNA dye) or via specific probes labeled with fluorescent dyes.

Molecular Taxonomy

Advantages Amplification is monitored in real-time against

standards of known concentration allowing for quantification of target DNA

When using specific probes for amplicon detection, the reaction can be multiplexed for simultaneous detection of up to 4 or 5 species and can be used on mixed samples

No electrophoresis is required, detection is automated and involves detection of fluorescence intensity

Allows for rapid and high throughput detectionDisadvantages Specialized equipment required Multiplexing is limited by choice of fluorescent dyes

Molecular Taxonomy

LAMP Loop-mediated isothermal amplification

Employs 3 sets of specific primers used for amplification under isothermal conditions.

Yields a ladder of amplicons on electrophoresis or amplicons can be detected using SYBR green

Molecular Taxonomy

Advantages Rapid and specific amplification under isothermal

conditions Technique is potentially the most suitable for field

conditions Can be used with mixed samples due to primer

specificityDisadvantages Assays have a relatively complex design Only suitable for field conditions when paired with a

simple DNA extraction method

Molecular Taxonomy

Reference Cheryl 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 Management, 3, 763-788, ISSN 2075-4450.

Sandhya Sukumaran and A. Gopalakrishnan , 2015 Molecular taxonomy – Applications, 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: Immunological distance.Evolution, :473-487.

Molecular Taxonomy

Thank you

Molecular Taxonomy