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BBIIOOCCHHEEMMIICCAALL 
MMAARRKKEERRSS 
Siddhartha Swarup Jena 
Ph.D. Mol. Bio & Biotech 
ANGRAU, Hyderabad
WWhhaatt iiss mmaarrkkeerr?? 
 Marker- that mark, tag or identify 
 Marker can be Morphological marker (size, 
shape, co...
MMaarrkkeerr -- WWhhaatt ffoorr?? 
 A breeder aims to improve the 
resistance of a cultivated form. 
Therefore, he/she pe...
TThhee ‘‘ppeerrffeecctt mmaarrkkeerr’’ 
• Polymorphic 
• Reproducible 
• Easy to use and economical 
• High-throughput 
– ...
MMaarrkkeerr ttyyppeess 
Isozymes, sec. metabolites 
S S JENA
BBiioocchheemmiiccaall mmaarrkkeerrss 
• Biochemical markers are proteins produced 
by gene expression. 
• Isozymes, alloz...
CCoommmmoonn pprrootteeiinn mmaarrkkeerrss-- IIssoozzyymmeess 
Isozymes were first described by R. L. Hunter 
and Clement ...
IIssoozzyymmeess 
Multiple forms of the same enzyme sharing a 
catalytic activity derived from a tissue of single 
organis...
S S JENA
IIssoozzyymmeess 
• To be useful as markers, isoforms must be 
electrophoretically resolvable, and detectable by gel 
assa...
MMeetthhooddoollooggyy 
• Grind and extract protein from appropriate tissue with 
buffer. 
• Fractionate the extract elect...
NNeecceessssaarryy hhaarrddwwaarree...... 
Electrophoresis 
electrophoresis 
assembly 
S S JENA
AAddvvaannttaaggeess ooff iissoozzyymmee mmaarrkkeerrss 
 They are not affected by the field or greenhouse 
environment. ...
LLiimmiittaattiioonnss ooff iissoozzyymmee mmaarrkkeerrss 
• Limited to those enzymes that can be detected in 
situ i.e. t...
IISSOOZZYYMMEE SSTTUUDDIIEESS IINN RRIICCEE 
• Chromosome Location of Isozyme Genes 
Enzyme Locus Chromosome 
Acid phospha...
IISSOOZZYYMMEE SSTTUUDDIIEESS IINN RRIICCEE 
Linkage of Isozyme Genes to Rice Traits 
• If the gene of interest is recessi...
The use ooff IIssoozzyymmeess iinn RRiiccee BBrreeeeddiinngg 
As biochemical marker, isozyme can be used for 
 germplasm ...
PPrrootteeiinn aalllloozzyymmeess 
• Electrophoretic variants of proteins produced by 
different alleles at protein-coding...
SSTTOORRAAGGEE PPRROOTTEEIINN MMAARRKKEERRSS 
• Storage proteins are a group of proteins stored in 
the seed that serve as...
LLeegguummiinn 
• Usually polymers with molecular weights around 
300,000 to 400,000 
• They are typically constructed fro...
VViicceelliinn 
• Vicelins are glycoproteins with 1-5% neutral sugar 
residues. 
• They, too, are constructed from several...
PPrroollaammiinneess 
• Prolamines, the storage proteins of gramineae, are 
characterized by a high proportion of proline ...
WWhhyy sseeeedd ssttoorraaggee pprrootteeiinnss?? 
• Seeds are a rich source of 
stable and abundant proteins 
• Seeds rep...
AAddvvaannttaaggeess ooff bbiioocchheemmiiccaall mmaarrkkeerrss 
 Inexpensive 
 Markers are co-dominant and allows heter...
DDiissaaddvvaannttaaggeess ooff bbiioocchheemmiiccaall mmaarrkkeerrss 
 Only reveals small proportion of DNA variation. 
...
UUsseess ooff MMaarrkkeerrss 
• Phenotyping and genotyping 
• Evolutionary relatedness 
• Contamination detection 
• Disea...
THANK YOU 
S S JENA
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Biochemical marker @ sid

Biochemical markers presentation

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Biochemical marker @ sid

  1. 1. BBIIOOCCHHEEMMIICCAALL MMAARRKKEERRSS Siddhartha Swarup Jena Ph.D. Mol. Bio & Biotech ANGRAU, Hyderabad
  2. 2. WWhhaatt iiss mmaarrkkeerr??  Marker- that mark, tag or identify  Marker can be Morphological marker (size, shape, color etc.), Biochemical markers (isozymes, proteins) and Genetic markers (DNA level)  Genetic marker- an allelic difference / variation at a given locus in the genome that can be observed at the level of morphology S S JENA
  3. 3. MMaarrkkeerr -- WWhhaatt ffoorr??  A breeder aims to improve the resistance of a cultivated form. Therefore, he/she performs a cross between the susceptible cultivated form with a wild form that possess the required resistance.  However, at least 6 backcrossing steps are necessary and the resistance is difficult to detect. S S JENA
  4. 4. TThhee ‘‘ppeerrffeecctt mmaarrkkeerr’’ • Polymorphic • Reproducible • Easy to use and economical • High-throughput – automation – combination of different markers in one reaction S S JENA
  5. 5. MMaarrkkeerr ttyyppeess Isozymes, sec. metabolites S S JENA
  6. 6. BBiioocchheemmiiccaall mmaarrkkeerrss • Biochemical markers are proteins produced by gene expression. • Isozymes, allozymes, proteins and secondary metabolites are successful biochemical markers. • First true molecular markers – derive their name from allelic variation of enzymes- Pyruvate dehydrogenase, Esterase, Peroxidase etc. S S JENA
  7. 7. CCoommmmoonn pprrootteeiinn mmaarrkkeerrss-- IIssoozzyymmeess Isozymes were first described by R. L. Hunter and Clement Markert (1957) who defined them as different variants of the same enzyme having identical functions and present in the same individual. Isozymes are usually the result of gene duplication, but can also arise from polyploidisation or nucleic acid hybridization. Tanksley and Rick (1980) used isozyme markers for the first time to speed up introgression of a monogenic trait into adapted tomato cultivars. S S JENA
  8. 8. IIssoozzyymmeess Multiple forms of the same enzyme sharing a catalytic activity derived from a tissue of single organism. Major functions of isozymes are controlling metabolic activities of the organism. Coded by same allele at more than one gene locus. (gene duplication; gene families) Allozyme: enzyme that is coded by different alleles at one gene locus Isozymes are generally codominant. S S JENA
  9. 9. S S JENA
  10. 10. IIssoozzyymmeess • To be useful as markers, isoforms must be electrophoretically resolvable, and detectable by gel assay methods. • The differences in size, configuration, and ionic charges among the isozymes allow them to be detected and resolved by various separation procedures. • Protein / Enzyme polymorphism can be revealed on electrophoregrams through a colored reaction associated with enzymatic activity. They are the products of various alleles of one or several genes. • Isozyme variation revealed in characteristic banding patterns is called zymogram. S S JENA
  11. 11. MMeetthhooddoollooggyy • Grind and extract protein from appropriate tissue with buffer. • Fractionate the extract electrophoretically in starch or polyacrylamide gels (non-denaturing) • Detect enzyme by incubation of gel (or gel print) in a solution of a synthetic substrate that allows the enzyme to catalyze a reaction that generates a colored product. S S JENA
  12. 12. NNeecceessssaarryy hhaarrddwwaarree...... Electrophoresis electrophoresis assembly S S JENA
  13. 13. AAddvvaannttaaggeess ooff iissoozzyymmee mmaarrkkeerrss  They are not affected by the field or greenhouse environment.  They are cost effective compared to other methods and the turnaround time is relatively rapid.  It is possible to identify outcrosses in inbred lines and selfs and outcrosses in single-cross and multi-cross hybrid populations with a very high level of precision and at a relatively low cost. S S JENA
  14. 14. LLiimmiittaattiioonnss ooff iissoozzyymmee mmaarrkkeerrss • Limited to those enzymes that can be detected in situ i.e. thin coverage of the genome. • Dimeric and multimeric enzymes add complexity. • Pattern can be influenced by environment and tissue-type specific. • Limited enzyme systems available (e.g. 15–20), limited no. of loci that can be scored (30- Tanksley & Orton, 1983). • The enzymes are not active in all the organs and all the stages. Not all of the reagent systems work efficiently with all plant species. S S JENA
  15. 15. IISSOOZZYYMMEE SSTTUUDDIIEESS IINN RRIICCEE • Chromosome Location of Isozyme Genes Enzyme Locus Chromosome Acid phosphatase Acp-1 Acp-2 3 6 Alcohol dehydrogenase Adh-1 11 Aminopeptidase Amp-1 Amp-2 Amp-3 Amp-4 2 8 3 8 Catalase Cat-1 3 Esterase Est-2 Est-4 Est-5 Est-8 3 1 1 7 S S JENA
  16. 16. IISSOOZZYYMMEE SSTTUUDDIIEESS IINN RRIICCEE Linkage of Isozyme Genes to Rice Traits • If the gene of interest is recessive, isozyme-based selection is particularly useful because the recessive gene can be followed without having to do progeny test. • Once linkage of the individual genes to isozyme markers is established, progeny testing to distinguish among individuals having the same phenotype but different numbers of independent genes controlling the character, may be omitted. S S JENA
  17. 17. The use ooff IIssoozzyymmeess iinn RRiiccee BBrreeeeddiinngg As biochemical marker, isozyme can be used for  germplasm classification,  gene mapping,  selection,  monitoring genetic segregation and recombination in distance crosses,  characterization F1 hybrid  nature of calli and somatic hybrid plants,  identification of sexual hybrid,  identification of plants from pollen or anther wall,  variety/hybrid purity, and  determination of phylogenic relationship in plant. S S JENA
  18. 18. PPrrootteeiinn aalllloozzyymmeess • Electrophoretic variants of proteins produced by different alleles at protein-coding genes. Protein Electrophoresis Gel • Alloenzymes are good markers for studies of population genetics, isoenzymes for development physiological ones. S S JENA
  19. 19. SSTTOORRAAGGEE PPRROOTTEEIINN MMAARRKKEERRSS • Storage proteins are a group of proteins stored in the seed that serve as nitrogen sources for the developing embryo during germination. • The proteins have no enzymatic activities • Legumes contain mostly two types of storage proteins- legumin and vicelin • Gramineae contain a third type: prolamin zeins (from Zea mays), hordeines (from Hordeum vulgare) etc. S S JENA
  20. 20. LLeegguummiinn • Usually polymers with molecular weights around 300,000 to 400,000 • They are typically constructed from two subunits, the acidic and the basic polypeptides. • The quaternary structure is composed of six acidic and six basic polypeptides that are linked by disulfide bonds. S S JENA
  21. 21. VViicceelliinn • Vicelins are glycoproteins with 1-5% neutral sugar residues. • They, too, are constructed from several polypeptide chains that are united by a quaternary structure. • All chains are cleavage products of one primary gene product. S S JENA
  22. 22. PPrroollaammiinneess • Prolamines, the storage proteins of gramineae, are characterized by a high proportion of proline and glutamine. They are soluble in alcohol. • They do also have a signal sequence but in contrast to legumines and vicelins, the polypeptide chain is not cleaved. • They are the products of a number of around 30-100 genes. Contrary to the genes of the leguminous storage proteins, they do not contain introns, i.e. each gene consists of one undivided translated DNA chain. • Among the best-known prolamines are the zeines, the storage proteins of maize. S S JENA
  23. 23. WWhhyy sseeeedd ssttoorraaggee pprrootteeiinnss?? • Seeds are a rich source of stable and abundant proteins • Seeds represent a well-defined developmental stage • Seeds are easily stored and transported • Each protein band in an electrophoretic profile usually represents a direct gene product SDS-PAGE of four wheat varieties S S JENA
  24. 24. AAddvvaannttaaggeess ooff bbiioocchheemmiiccaall mmaarrkkeerrss  Inexpensive  Markers are co-dominant and allows heterozygous to be distin-guished from homozygous.  Iso-zymes rarely exhibit epistatic interaction so that a genetic stock containing an infinite number of markers could be constructed.  The process is nondestructive since only small amounts of plant tissue are needed.  Any plant tissue can be used as samples, including leaves, roots, pollen, and callus, so that the technique is very versatile. S S JENA
  25. 25. DDiissaaddvvaannttaaggeess ooff bbiioocchheemmiiccaall mmaarrkkeerrss  Only reveals small proportion of DNA variation.  Many DNA variants do not result in changes in amino acid sequence (e.g., synonymous substitutions).  Some changes in amino acid sequence do not result in changes in mobility on the gel.  Many different biochemical procedures are required. S S JENA
  26. 26. UUsseess ooff MMaarrkkeerrss • Phenotyping and genotyping • Evolutionary relatedness • Contamination detection • Disease diagnosis • Forensic evidence • Marker assisted breeding S S JENA
  27. 27. THANK YOU S S JENA

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