Molecular taxonomy

presentation gives you information about different techniques and markers used in molecular taxonomy along with its advantages and disadvantages

1. MOLECULAR TAXONOMY Submitted by- M.Illakeya I M.Sc PHE Molecular Taxonomy

2. Taxonomic Characters Morphological, Cytological, Ethological, Ecological and Biochemical characters. Molecular Taxonomy

3. 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 )

4. 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

5. 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

6. 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

7. Molecular markers Molecular markers can be characterized as Type I and Type II markers; Type I markers are associated with genes of known function and T ype 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

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 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

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 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

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. 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

11. 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

12. 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

13. 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

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

15. 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

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 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

17. Size differential PCR Employs generic PCR primers but yields amplicons that differ in length. Usually targets the intergenic transcribed spacer regions (ITS). Molecular Taxonomy

18. 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

19. PCR-RFLP Involves discrimination of species based on restriction profile of amplicons. Molecular Taxonomy

20. 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

21. Multiplex PCR Combines multiple primer sets with different specificities in a single assay Molecular Taxonomy

22. 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

23. RAPD Uses random primers to generate multiple PCR products resulting in a fingerprint for a particular species. Molecular Taxonomy

24. 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

25. AFLP Involves ligation of adaptors to digested DNA followed by PCR amplification using primers that are partially adaptor and partially gene-specific Molecular Taxonomy

26. 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 instances Disadvantages 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

27. Microsatellite analysis Involves PCR amplification of multiple reiterated repeat-containing loci that are hypervariable due to slipped-strand mispairing mutations Molecular Taxonomy

28. Molecular Taxonomy

29. 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

30. 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

31. 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

32. 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

33. 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

34. 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

35. Thank you Molecular Taxonomy