Biochemical markers presentation

1. BBIIOOCCHHEEMMIICCAALL
MMAARRKKEERRSS
Siddhartha Swarup Jena
Ph.D. Mol. Bio & Biotech
ANGRAU, Hyderabad

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. 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.
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4. TThhee ‘‘ppeerrffeecctt mmaarrkkeerr’’
• Polymorphic
• Reproducible
• Easy to use and economical
• High-throughput
– automation
– combination of different markers in one
reaction
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5. MMaarrkkeerr ttyyppeess
Isozymes, sec. metabolites
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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.
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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.
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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.
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9. S S JENA

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.
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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.
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12. NNeecceessssaarryy hhaarrddwwaarree......
Electrophoresis
electrophoresis
assembly
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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.
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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.
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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
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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.
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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.
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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. 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.
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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.
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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.
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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.
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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. 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.
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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.
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26. UUsseess ooff MMaarrkkeerrss
• Phenotyping and genotyping
• Evolutionary relatedness
• Contamination detection
• Disease diagnosis
• Forensic evidence
• Marker assisted breeding
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27. THANK YOU
S S JENA