Molecular taxonomy

Submitted by-
M.Illakeya
I 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 analyses
Disadvantages
 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 species
Advantages
 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 required
Disadvantages
 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 instances
Disadvantages
 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 instances
Disadvantages
 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
or cryptic species
 Very sensitive and more robust than RAPD
 Data may be used for phylogenetic reconstruction in
some instances
Disadvantages
 Requires manipulations in addition to PCR
 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

Advantages
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
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 detection
Disadvantages
 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
specificity
Disadvantages
 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

Molecular taxonomy

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Molecular taxonomy