Simple sequence repeat (SSR) markers consist of di- or tri-nucleotide repeats and have many advantages as genetic markers. They display high diversity and have many possible alleles per locus. While SSR markers are useful, single nucleotide polymorphism (SNP) markers are becoming more widely used due to their greater reliability and cost-effectiveness. Microsatellites can be amplified using PCR to analyze relatedness and identify individuals, but they have limitations such as null alleles occurring when primers don't correctly amplify loci.

1. Discussion
5.1 SSR markers:
SimpleSequenceRepeats (SSR), Simple Tandem Repeats (STRs)
or Microsatellites markers are usually consist of di or tri-nucleotide
repeats {ATGATGATG is an example of a tri-nucleotide repeat (ATG)}.
These markers can be relatively efficient if more than one primer set is
combined (multiplexing) in a single reaction. SSRs also have the
advantage of displaying great diversity. SSR/STR markers have the
advantage of having a large number of alleles for a given locus (marker).
This is because there is really no limit to the number of repeats that can
occur. In some cases (e.g. maize) it is possible to have 20 unique alleles
for one marker. This information is very helpful as it provides an efficient
way to track alleles in a population and discriminate between several
individuals using a relatively small number of markers.DNA Land Marks
has been working with SSR/STR markers for many years, but in the past
few years has been transitioning its SSR/STR marker work to SNP
markers. SNP markers offer many benefits over SSR/STR markers,
including greater reliability of results and better cost effectiveness. More
and more research is being done on SNP markers, leading to the
discovery of these markers in an ever increasing number of species.
5.2 Analysis of microsatellites:
Microsatellites are widely used for DNA
profiling in kinship analysis (most commonly in paternity testing) and in
forensic identification (typically matching a crime stain to a victim or
perpetrator). Also, microsatellites are used for mapping locations within
the genome, specifically in genetic linkage analysis/marker assisted

2. selection to locate a gene or a mutation responsible for a given trait or
disease. As a special case of mapping, they can be used for studies
of gene duplication or deletion. These applications are realized by various
technical methods.
5.3 Amplification:
Microsatellites can be amplified for identification by
the polymerase chain reaction (PCR) process, using the unique sequences
of flanking regions as primers. DNA is repeatedly denatured at a high
temperature to separate the double strand then cooled to
allow annealing of primers and the extension of nucleotide sequences
through the microsatellite. This process results in production of enough
DNA to be visible on agarose or polyacrylamide gels; only small amounts
of DNA are needed for amplification because in this way thermocycling
creates an exponential increase in the replicated segment. With the
abundance of PCR technology, primers that flank microsatellite loci are
simple and quick to use, but the development of correctly functioning
primers is often a tedious and costly process.
5.4 Limitations:
Microsatellites have proved to be versatile molecular markers,
particularly for population analysis, but they are not without limitations.
Microsatellites developed for particular species can often be applied to
closely related species, but the percentage of loci that successfully
amplify may decrease with increasing genetic distance. Point mutation in
the primer annealing sites in such species may lead to the occurrence of
‘null alleles’, where microsatellites fail to amplify in PCR assays. Null
alleles can be attributed to several phenomena. Sequence divergence in

3. flanking regions can lead to poor primer annealing, especially at the 3’
section, where extension commences; preferential amplification of
particular size alleles due to the competitive nature of PCR can lead
to heterozygous individuals being scored for homo zygosity (partial null).
PCR failure may result when particular loci fail to amplify, whereas
others amplify more efficiently and may appear homozygous on a gel
assay, when they are in reality heterozygous in the genome. Null alleles
complicate the interpretation of microsatellite allele frequencies and thus
make estimates of relatedness faulty. Furthermore, stochastic effects of
sampling that occurs during mating may change allele frequencies in a
way that is very similar to the effect of null alleles; an excessive
frequency of homozygotes causing deviations from Hardy-Weinberg
equilibrium expectations. Since null alleles are a technical problem and
sampling effects that occur during mating are a real biological property of
a population, it is often very important to distinguish between them if
excess homozygotes are observed.
When using microsatellites to compare species, homologous loci may be
easily amplified in related species, but the number of loci that amplify
successfully during PCR may decrease with increased genetic distance
between the species in question. Mutation in microsatellite alleles is
biased in the sense that larger alleles contain more bases, and are
therefore likely to be mistranslated in DNA replication. Smaller alleles
also tend to increase in size, whereas larger alleles tend to decrease in
size, as they may be subject to an upper size limit; this constraint has
been determined but possible values have not yet been specified. If there
is a large size difference between individual alleles, then there may be
increased instability during recombination at meiosis. In tumor cells,
where controls on replication may be damaged, microsatellites may be

4. gained or lost at an especially high frequency during each round
of mitosis. Hence a tumor cell line might show a different genetic
fingerprint from that of the host tissue.
5.5 Justification:
1. Chromosomes were so densely packed structures in all organisms but
some cases they were relaxed and some cases twisted form. In
nucleosome model Chromosome was much condensed form so in
Sorghum bicolor.
2. Sorghum Chromosome 1(CM000760.1) size was much 73840631bp
while SSRs number is 15676 that there were junk form of sequence
,Which may express either in stress condition(abiotic/biotic).
3. Repeating units in a any highly variable, characteristic that makes them
useful as genetic marker.
4. Variation in the number of repeats has no consequence on gene
function. Generally the microsatellite itself does not cause disease, but
rather is used as a marker to identify a specific chromosome or locus.
5. SSR marker motifs (AT) contains markers with an alternative highly
sensitive method could be used for polymorphism detection (higher level
of diversity.