Markers for genetic mapping; methods and

techniques used for genetic mapping

Dr. Jatindra Nath Mohanty

Jatindranath.mohanty@cutm.ac.in
How It Works
• Unlike traditional PCR analysis, RAPD (pronounced "rapid") does not
require any specific knowledge of the DNA sequence of the target
organism: the identical 10-mer primers will or will not amplify a
segment of DNA, depending on positions that are complementary to
the primers' sequence. For example, no fragment is produced if
primers annealed too far apart or 3' ends of the primers are not
facing each other.
• Therefore, if a mutation has occurred in the template DNA at the site
that was previously complementary to the primer, a PCR product will
not be produced, resulting in a different pattern of amplified DNA
segments on the gel
Developing Locus-specific, Co-Dominant Markers from RAPDs
• The polymorphic RAPD marker band is isolated from the gel.
• It is amplified in the PCR reaction.
• The PCR product is cloned and sequenced.
• New longer and specific primers are designed for the DNA
sequence, which is called the Sequenced Characterized Amplified
Region Marker (SCAR).
 ISSR marker (Inter Simple Sequence Repeats)

• ISSRs are DNA fragments of about 100-3000 bp located between

adjacent, oppositely oriented microsatellite regions.
• ISSRs are amplified by PCR using microsatellite core sequences as
primers with a few selective nucleotides as anchors into the non-repeat
adjacent regions (16-18 bp).
• About 10-60 fragments from multiple loci are generated
simultaneously, separated by gel electrophoresis and scored as the
presence or absence of fragments of particular size. Techniques related
to ISSR analysis are Single Primer Amplification Reaction (SPAR) that
uses a single primer containing only the core motif of a microsatellite
 Annealing Temp
Primer code Name of Primer Sequence (5’-3’) No. of bands amplified
(in 0C)
ISSR1 T(GA)9 TGAGAGAGAGAGAGAGAGA 51 10
ISSR 2 (GTGC)4 GTGCGTGCGTGCGTGC 51 11
ISSR3 (GA)9 T GAGAGAGAGAGAGAGAGAT 51 9
ISSR4 (GACA)4 GACAGACAGACAGACA 43 7
ISSR5 (GAC)5 GACGACGACGACGAC 45 6
ISSR6 (GTG)5 GTGGTGGTGGTGGTG 45 8
ISSR 7 (AGG)6 AGGAGGAGGAGGAGGAGG 55 8
ISSR8 (CAA)5 CAACAACAACAACAA 36 7
ISSR9 (GA)8 C GAGAGAGAGAGAGAGAC 47 10
ISSR10 (GA)8 A GAGAGAGAGAGAGAGAA 45 7
ISSR11 (CT)8 T CTCTCTCTCTCTCTCTT 45 9
ISSR 12 (CT)8 A CTCTCTCTCTCTCTCTA 45 6
ISSR13 (CT)8 G CTCTCTCTCTCTCTCTG 47 9
ISSR14 (CA)8 T CACACACACACACACAT 45 8
ISSR15 (CA)8 A CACACACACACACACAA 46 10
ISSR16 (CA)8 G CACACACACACACACAG 47 13
ISSR 17 (AT)8 C ATATATATATATATATC 36 5
ISSR18 (TA)8 A TATATATATATATATAA 30 Smear
ISSR19 (TA)8 C TATATATATATATATAC 36 11
ISSR20 (TA)8 G TATATATATATATATAG 36 6
ISSR21 (AG)8 T AGAGAGAGAGAGAGAGT 50 5
ISSR 22 (GA)8 T GAGAGAGAGAGAGAGAT 50 8
ISSR23 (GT)8 A GTGTGTGTGTGTGTGTA 50 12
ISSR24 (AG)8 C AGAGAGAGAGAGAGAGC 47 13
ISSR25 (AG)8 G AGAGAGAGAGAGAGAGG 47 9
ISSR26 (AT)8 T ATATATATATATATATT 35 Smear
ISSR27 (AT)8 G ATATATATATATATATG 36 6
Total 213
Strengths
• The main advantage of ISSRs is that no sequence data for primer
construction are needed. Because the analytical procedures include
PCR, only low quantities of template DNA are required.
• Furthermore, ISSRs are randomly distributed throughout the
genome.
Weaknesses
• Because ISSR is a multilocus technique, disadvantages include the
possible non-homology of similar sized fragments. Moreover,
ISSRs, like RAPDs, can have reproducibility problems.
Applications

• Because of the multilocus fingerprinting profiles obtained, ISSR

analysis can be applied in studies involving genetic identity,
parentage, clone and strain identification, and taxonomic studies of
closely related species. In addition, ISSRs are considered useful in
gene mapping studies.