6-5.3(a).1.5(ii) Microsatellite

Microsatellite also known as short tandem repeats (STRs) or simple sequences repeats (SSR) are short repeating nucleotides (2-6 nts). In plants di-,tri-, and tetra- nucleotide repeats like (GT)_n ,(AAT)_n , and (GATA)_n are found widely distributed. In fishes microsatellites have been estimated to occur once in every 10 kb (Wright, 1993) of DNA length. Copy number of these repeats varies from individual to individual and forms the basis of polymorphism. One of the illustrative examples of microsatellites within coding regions are those causing genetic diseases in humans, such as the CAG repeats that encode polyglutamine tract, resulting in mental retardation. Microsatellite markers are highly polymorphic, and occur in large numbers. It is co-dominant and has ability to distinguish multiple alleles in a plant. Two conserved and locus-specific PCR primers flanking each microsatellite repeat are used for PCR amplification.

Several detection methods have been developed based on primer labeling, gel utility, staining, and detection equipment. Another way to detect SSR products is carried out on a DNA sequencer that detects fluorescent dye labels by laser excitation.

6-5.3(a).1.6 Inter Simple Sequence Repeat (ISSR)

As name the suggest “inter simple sequence repeat” involves amplification of DNA regionthat is located in between the two identical microsatellite repeats that are oriented in opposite direction. Briefly ISSR analysis helps in the understanding of organization, frequency and levels of polymorphism of different SSR's in a genome (Reddy 2002). ISSR is a PCR based method where16–25 bp long microsatellites are used as primers in a single primer PCR reaction to target multiple genomic loci for the amplification of mainly the inter- SSR sequences of variable sizes.

The primers used are either unanchored (Gupta et al., 1994; Meyer et al., 1993) or anchored at 5'or 3' end with 1-4 degenerate bases extended into the flanking sequences (Zietkiewicz et al., 1994).

►ISSR associates most of the advantages of AFLP and microsatellite analysis with the universality of RAPD. However as longer primers are used as compared to RAPD primers (10- mers) deployment of high annealing temperature (45–60°C) provides higher reproducibility and stringency.

►ISSRs mostly segregate as dominant markers following simple Mendelian inheritance (Gupta et al., 1994) and in few cases as co-dominant markers. The latter enabling distinction between homozygotes and heterozygotes (Wu et al., 1994; Akagi et al., 1996).

The major areas of the application of ISSR are as below,

i. Genomic fingerprinting

ii. Genetic diversity and phylogenetic analysis

iii. Genome mapping

iv. Determining SSR motif frequency

v. Gene tagging and use in marker assisted selection

vi. Evolutionary biology.

There are many other markers which can be used for mapping in plants. Few of them explained above and the comparative study of the five most commonly used DNA markers are give in table 6-5.3(a).1.