MOLECULAR MARKERS

1. MOLECULAR MARKERS

2. Definitions:- • Marker:-constituent that determines the function of a construction • Genetics marker:- • Any stable & inherited variation • that can be measured or detected by a suitable method & • can be subsequently used to detect the presence of a specific genotype or phenotype other than itself, which otherwise is non measurable or difficult to detect • It is an indirect way of predicting variation.

3. Types of Genetic Markers • Phenotypic markers (morphological markers) • Chromosomal markers • Biochemical markers • Molecular markers

4. Dominant Allele: An allele that expresses its phenotypic effect even when heterozygous with a recessive allele; thus if A is dominant over a, then AA and Aa have the same phenotype. • Codominant Allele: an allele that shows codominance (both alleles are separately apparent in the phenotype)

5. Phenotypic markers:- phenotype for which the variation observed in the population of interest is entirely explained by Mendelian factor • Eg. : height , color, polled or horned phenotype in cattle etc…….. • Limitations: -Show low degree of polymorphism -Reflect variability in the coding sequence only

6. Chromosomal markers:- • Structural & numerical variations of chromosomes • Limitations: • Low degree of polymorphism

7. Biochemical markers:- • Observed in the form of macromolecule present in the body fluids (milk , urine,blood,etc…) & tissues • are detectable immunological (blood groups, MHC etc..) & Electrophoretic methods (serum protein milk protein etc…) • Limitations: • Age dependent, sex limited, influenced by the environment • Reflect variability in the coding sequence only

8. Molecular markers: • Variations at DNA sequence level • Basic Advantages • detect variation at coding as well as non-coding sequences • High degree polymorphism • Usually express in co-dominant fashion or in dominant fashion (less preferred) • Multi-allelic • Unaffected by the environmental factors • Locus specific • Numerous & distributed ubiquitously throughout the genome

9. Methodological advantages of molecular markers: • Require DNA samples which can isolated from blood , tissues, sperm, hair follicle etc….. • DNA samples can stored • Analysis can carried out even at embryonic stage • Repeated experiments with same DNA possible once it transferred to solid phase like filter membranes • PCR methods can be applied & so can subjected to automation

10. Molecular markers • RFLP markers ( restriction fragment length polymorphism) • RAPD markers • AFLP markers • Minisatellite markers (VNTR) • Micro satellites • SNP

12. RFLP markers

13. RFLP Allele A Allele B

14. Carrier identification

15. Applications • Identification of disease carrier • Parentage determination • Advantages • Co-dominant marker • High reproducibility • Limitations • High quantity DNA required • Should know the sequence • Moderately polymorphic(SLDA) • Laborious & technically demanding

16. PCR-RFLP (CAPS)

17. RFLP have been reported at Beta globulin locus in pig (Rando,A. Marina,P.) MHC locus in cattle , pig , sheep ,horse (Vaiman,M. Chardon,P. Cohen,D. (1986)

18. Dominant and Codominant Markers Dominant markers: viz. RAPDs and AFLPs and SMPLs allow the analysis of many loci per experiment without requiring previous information about their sequence. Codominant markers: viz. RFLPs, microsatellites etc. allow the analysis of only a locus per experiment, are more informative because the allelic variations of that locus can be distinguished. One can identify linkage groups between different genetic maps For their development it is précised to know the sequence

19. RAPD (Random amplified polymorphic DNA)

20. Application • Genetic distance estimation Advantages • Easily generated • Sequence information not needed Disadvantages • Less reproducible • Dominant marker • New map be generated for each new pedigree

21. AFLP(Amplified fragment length polymorphism)

22. Restriction digest genomic DNA – double digest (e.g. EcoRI and MseI) – leaves fragments with different sticky ends. Ligate linker to fragments--  Each end has a different linker – complementary to sticky ends left by restriction enzyme.  The linkers have unique and known sequences that can be used to build primers. Conduct a preselective PCR amplification using primers that are complementary to the two linkers and that have one additional base extended from their 3’ ends.  This lowers the pool of fragments that will amplify.  The primer with the additional base can be made with an extension step in a thermal cycler – OR – just purchased. Conduct a second PCR amplification using additional nucleotide(s) at the 3’ ends of the linker primers.  This reduces the pool of fragments even more.e. Run acrylamide gel and visualize the bands.

24. DFP( DNA finger printing) • VNTR ( Variable number tandem repeats) –coined by Nakamura to describe individual loci where alleles are composed of tandem repeats that vary in number of core units. • VNTR of two animal may be of same length & sequence at certain site , but vary at other sites • As originally coined , DFP , refers to the multiband print produced using multilocus multiallele minisatellite probes.

25. PRINCIPLE

26. Applications • Parentage determination • Animal & plant’s economic trait characterization • Investigation in forensic science • wild life paternity • Individual identification • Genetic distance estimation • Gene mapping

27. Microsatellite • Hybridization based • PCR based Uses • Parentage determination • Genetic diversity studies • Gene mapping Advantages • Hyper variability • Co-dominant marker • Distributed all over genome • Easiness of detection, reliable • number of microsatellite can be analysed at a time

29. SNP (Single nucleotide polymorphism) • May due to nucleotide substitution (transition, transvertions) or insertion , deletion • It is found that SNP occurs at a rate of 1 in 1000 bp in human. (Cooper et al 1985) • Richest source of variation

30. SSCP(single stranded conformation polymorphism) Principle • Migration of ssDNA through gel depends on the sequence and the size . • dsDNA ssDNA PAGE (SSCP) Advantages • Relatively simple • Low cost equipment • Detect SNP at multiple site & also at site where no restriction site involved Formamide Methyl mercuro chloride

31. Applications • To understand mechanism of diseases • Analyze the mutation Limitation • Accuracy is 65-70 percent • Accuracy depends on various physical factors and need to be optimized • Detection sensitivity reduces as the size increases (esp >200bp)

32. APPLICATIONS

33. Applications in conventional breeding strategies Short range applications • parentage determination • Genetic distance estimation • sex determination • Identification of carrier Long range applications • Gene mapping • Marker assisted selection

34. Parentage determination • For the estimation of breeding value • Confirmation of parent in IVF calves • Verification of semen • Animal identification

35. Genetic distance estimation • Is measure of evolutionary divergence • Characterization of different breeds • Authentication of pedigree • Evaluation of change in the variation in a species over time

36. Sex determination • For sexing of pre-implantation embryo Other technologies • Invasive-FISH, Y-probe, cytogenetic method, PCR method • Noninvasive- HY-antigen, X linked enzymes

37. Identification of disease carrier • It is found that certain allelic variations in the host genome leads to susceptibility or resistance to a particular disease (Kingsbury,D.T.1990) • Using microsatellite (TGLA116) marker George et al demonstrated the identification of carrier allele of weaver disease in cattle (1993)

38. Long range applications • Gene mapping • Marker assisted selection

39. Gene mapping Genetic map: are sets of loci arranged in order & separated by distance( in unit depending on type of map) with each set corresponding to a chromosomes pair (a)Physical map (with help of molecular tech) • Somatic cell hybrid • In situ hybridization • Comparative mapping • Radiation map (b) genetic map (based on the recombination frequency) • Linkage map

40. Applications • A molecular marker allow direct identification of gene of interest,serves as a useful tool for screening somatic cell hybrid • Physical mapping become easy • Provide sufficient markers for construction map using linkage analysis Markers used • Type I markers –RFLP markers • Type II markers- microsatellite Uses of map • Used for population studies • Tracking the inheritance of DNA • Parentage identification etc…….

41. Marker assisted selection • When genetic marker data are included in the selection criteria, this process being referred to as marker-assisted selection . Applications • MAS is likely to compliment rather than replace conventional breeding system • Genetic improvement in through - higher selection intensity - reduction in generation interval - increase accuracy of prediction - selection possible in early life - sex-limited & carcass trait

42. Limitations • Family specific nature of information • Size and complexity of genetic formulation • statistical methodology • The availability of limited number of markers is also the limitation of this methodology • As the total genomic map is not established, it is difficult to know the position and structure of genes in QTL • The requirement of large number of offspring for linkage analysis is also another limitation.

43. Transgenic breeding system • It serves as reference point for mapping the relevant genes • Identification of animals carrying the transgene

44. Other Applications • Identification of genes responsible for diseases resistance & their introgression • egs: gene for trypanosoma tolerance from N’Dama cattle • Booroola gene for increase fecundity

45. Constraints • Lack of sufficient polymorphic probes for large animals • In MAS the information regarding linkage should be accurate otherwise negative results occur.

46. Conclusion • Many types of molecular markers available • Type(s) chosen for use will depend on many factors • Dominant or co-dominant, co-dominant preferable • All molecular markers are not equal. None is ideal. Some are better for some purposes than others. However, all are generally preferable to morphological markers for mapping.