INTRODUCTION

Today’s era is considered to be the era of genetic revolution which makes the use
of polymorphic genes as the molecular markers to bring the green revolution few steps
forward in the field of crop-breeding programmes. Exploitation of genetic resources
have been a routine work for the scientists to discover the agronomic traits of interest in
plants.For this extensive studies in plants are being done. Moreover the versatile
techniques and instruments in the field of molecular biology making such tough tasks to
look more easier for getting the best and reproducible results.As Maize is one of the the
most economical crop, vast studies has been going on using such polymorohic genes.

Maize is considered to be one of the most economical plant from its nutritional
point of view also from genetic point of view.It is the third largest cultivated crop in
India.The maize (Zea mays L.) is a monoecious annual plant which belongs to maideas
tribe and the grass family of Gramineae and their cells have 2n chromosomes.It is the
only cereal, which is grown systematically by American & Indians. Christopher Colombo
encounters that maize was cultivated in Haiti, where it was named "Mahiz". He carried
the maize from America to Europe and later was carried by Portuguese and others
Europeans to Africa and Asia, during 16th and 17th centuries. The Gramineae is
comprised or approximately 120 genera and 3100 species distributed In both tropical and
temperate regions. Central and south America are the major areas of distribution where
over 55 genera are found, About 23 genera and over 85 species have been reported from
India. Some of the larger genera along with their approximately world over reported
species (Hickey and Steward1981) . The maize is the most domesticated and evolutioned
plant of the vegetal kingdom, however the origin and evolution of the maize is a mystery,
since it has arrived to us highly evolutioned without intermediate forms, while the cereals
from the old continent have intermediate wild varieties which are identified and
preserved by nature. Notwithstanding, since the 19th century diverse theories have been
exposed to explain the origin and evolution of the maize, of which one of the most
accepted is that the direct predecessor of the maize is the Teosintle.

The plant of maize has distichous leaves (two ranks of single leaves borne in
alternate position). The leaf blades tend to be held at right angles to the sun by stiff mid-
ribs. The external surface of the leave blade is adapted for the absorption of solar energy
by little hairy structures and the internal surface is shiny and hairless with numerous
stomata for breathing.The maize plant, exhibits a single predominant stem with some few
basal branches (tillers), they serve as feeder for the root system. Longer tillers may
compete with the main stem and tillers of intermediate length may have terminals
inflorescences which are structurally intermediate between tassels (male inflorescence)
and ears (female inflorescences). The male inflorescence terminates on the uppermost
spike branched arranged in a loose panicles. On this structure, the flower are organised
into paired spikelets into each spikelets and there are two functional florets and each one
has three anthers. kernels per ear.. Maize varies widely in height, normal average is 2.4
m. Maize is cultivated at latitudes 50 degrees north and south, and even slightly higher
from the Equator, also from sea level to 3600 meters elevation in cool and hot weathers,
and with growing cycles oscillating from 3 up to 13 months.It is a versatile crop, and it
has tremendous genetic variability,which enables it to thrive well under lowland tropical,
subtropical, and temperate climates. It is grown in more countries than any other cereal.
In the middle of 19th and beginning of 20th centuries respectively, Indian farmers and
seeds men developed outstanding open-pollinated varieties, and intensive research in
plant breeding offered spectacular improvement in crop yields. Hybrid maize is the
greatest practical achievement of plant genetics to date.

The maize represents to all maize-based groups a source of life. original from It is very
adaptable to different weathers, and nowadays its consumption is worldwide. In fact, the
maize is the most widely grown cereal crop. In the global production of cereals crops, the
maize rank first after rice (paddy) and wheat. Likewise, in countries with developing
economies, such as Latin-American and Africa the maize rank first. In Asia rank third
after rice and wheat.

The presence of highly polymorphic repetitive DNA (SSRs) in maize helps in the
selection of traits of interests which are linked to the SSRs. These are inherited according
to the Mandelian principle.These SSRs are scored through DNA amplification using
SSR-PCR ultimately run in a agarose-gel system.Generally these SSR-PCR uses touch-
down pcr profile making all the different primers with different annealing temp,
compatible to one PCR reaction.
POLYMERASE CHAIN REACTION:
PCR i.e. Polymerase chain Reaction is a biochemical and a molecular technique
for amplifying the target DNA sequence through the application of enzymatic replication
done in an in vitro condition by using an automated thermal cycler.It is a method of
exponential amplification of a given DNA template with the help of polymerase in an
automated instrument called as thermalcycler.

The basic principle of replicating a piece of DNA using two primers had already
been described by Gobind Khorana in 1971:– Kleppe et al. (1971) J. Mol. Biol. 56, 341-
346.But Progress was limited by primer synthesis and polymerase purification issues.
Mullis properly exploited amplification.PCR was invented by Kary Mullis in 1983.First
published account appeared in 1985.( Awarded NobelPrize for his contribution to
Chemistry in 1993.)

Time in Minute

Figure-1. showing a thermal cycler profile in a general PCR reaction

The potential of PCR to amplify a specific DNA sequence from a complex

template in an automated procedure has opened the doors for many molecular research
works.

The components of a PCR involve the following contents:

 A DNA template that contains the region of DNA fragments to be amplified.

 Set of both forward and reverse primers which are complimentary to the DNA at the
5’ and 3’ ends of the DNA which is to be amplified.

 A thermo -stable DNA polymerase (e.g.: Taq or Pfu polymerase having an optimum
temp of 700c for replication) for the synthesis of DNA copies from the region of
interest.

 Deoxynucleotide triphosphates (dATPs, dTTPs, dGTPS, dCTPs) with the help of

which the DNA polymerase synthesize new DNA strands. Now days vent polymerase
is also used having its own specific function. Among these Taq was first discovered
at yellow stone national park by Thomas Brock in the hot spring. It is being derived
from the bacteria Thermus aquaticus.

 A buffer solution which provides a suitable biochemical environment for the stability
and the efficient activities of a DNA polymerase. Usually Tris-accetate EDTA (TAE)
and TBE (Tris-borate EDTA) is used with concentration of 1x.

 A divalent cat-ion mostly Mg2+ which act as metallic co factor for the DNA
polymerase involved in PCR.

 A thermal cycler having wells for holding the reaction tubes of PCR. These tubes
having a volume of 25 to 100 µL of DNA. This machine having the facilities of
maintaining a specific temperature for a specific reaction during the amplification.
These thermal cyclers are provided with fitted lids to prevent the condensation of the
reaction tube caps or a layer of oil can be replaced on the reaction tube to prevent
evaporation. It is an automated instrument maintaining the required temperature.

figure-2 showing a PCR machine (BIO-RAD)

 PCR water is specifically designed water used for PCR. PCR water is nucleases free
and nonionic so that it remains inert to the reaction mixture. Water is added to make
up the final volume. The volume of the water is determined by adding up all the
volumes of constituent in a reaction mixture and their sum is subtracted by the final
volume.

 Additives are the agents which are added in order to enhance the PCR reaction.
dNTP’s, additives like gelation or BSA and non-ionic detergents like Tween-20 or
Nonidet P-40 or Triton X-100 are know to greatly enhance the reaction. DAMO,
DMF, Urea, Betaine, BSA, Gelatine Pyrophosphate, DMSO etc. Some enzymes do
not need added protein. Some enzymes work in the presence of detergent. Reaction
overlay- Mineral oil (Oil) or Paraffin (Tablet) whenever they are added the products
needs to be purified.The PCR involves a series of 20 to 35 cycles which are carried
out basically in three steps given below:

 Denaturation (Generally occurs at 950C-940C for 5-3 mins)

 Annealing (Generally occurs at 600C-500C for 2-3 mins)

 Extension (Generally occurs at 750C-650C for 2-1 mins)

Now a days looking at the purpose of the research works ande project many
modification are done in PCR such as allespcific PCR(SNP),Asymmetric PCR,Hot Start
PCR,Inverse PCR,ISSR-PCR,SSR-PCR,Touch-down PCR,Real time PCR,Reverse
transcription PCR,ALU-PCR.
Figure-3 showing an overview of a general PCR reaction
Simple Sequence Repeat(SSR):
Variations in DNA sequences have been explored as molecular markers for
mapping important genes in plants and animals, during the last two decades. With the
advent of the polymerase chain reaction (PCR), new classes of markers emerged that
combined the desired characteristics of being highly polymorphic and cost effective, such
as RAPD, AFLP, and, more recently, microsatellites or SSRs. The latter consist of 1- to
6-bp nucleotide motifs repeated 10 to 50 times in tandem. They can be amplified by
PCR, provided the non-repetitive sequences that flank them become known.

The term microsatellites was coined by Litt and Lutty2, are multilocus DNA
sequences creating complex banding patterns and are usually non-species specific
occurring ubiquitously. They essentially belong to the repetitive DNA family. SSRs)
consist of 1 to 6 bp long monomer sequence that is repeated several times. These loci
contain tandem repeats that vary in the number of repeat units between genotypes and are
referred to as variable number of tandem repeats (VNTRs) (i.e. a single locus that
contains variable number of tandem repeats between individuals27) or hypervariable
regions (HVRs) (i.e. numerous loci containing tandem repeats within a genome
generating high levels of polymorphism between individuals2). Microsatellites thus form
an ideal marker system creating complex banding patterns by simultaneously detecting
multiple DNA loci. The prominent features of the marker is that they are dominant
fingerprinting markers and co-dominant STMS fingerprinting markers (sequence tagged
microsatellites).

Microsatellites, or Simple Sequence Repeats (SSRs), are polymorphic loci present

in nuclear and organellar DNA that consist of repeating units of 1-6 base pairs in length.
They are typically neutral, co-dominant and are used as molecular markers which have
wide-ranging applications in the field of genetics, including kinship and population
studies. Microsatellites can also be used to study gene dosage (looking for duplications or
deletions of a particular genetic region).
A microsatellite consists of a specific sequence of DNA bases or nucleotides
which contains mono, di, tri, or tetra tandem repeats. Microsastellites are inherited in a
Mendelian fashion. For example,
AAAAAAAAAAA would be referred to as (A)11
GTGTGTGTGTGT would be referred to as (GT)6
CTGCTGCTGCTG would be referred to as (CTG)4
ACTCACTCACTCACTC would be referred to as (ACTC)4
SSR markers in specific regions of a genome are determined by taking the flanking
sequences ,which are so used to design oligonucleotide primers which will amplify the
specific microsatellite repeat in a PCR reaction.

Figure-4 showing an example of SSR(bnlg2234 ) in Maize with DNA repeat ofs 16bp
having the flanking sequences of 50bps.
PCR products are then generally resolved through capillary electrophoresis or high
resolution agarose gel electrophoresis.

Application of SSRs using SSR-PCR:

PCR using SSR loci (SSR-PCR) has got many applications as microsatellites are
widely dispersed in eukaryotic genomes, are highly variable, and are requiring only
minute amounts of starting template for genomic fingerprinting thus helpful in studying
genetic diversisity & genetic distances in plants .Genome fingerprints used in diagnosis
and identification of Human Diseases as microsatellites change in length early in the
development of some cancers, they are useful markers for early cancer detection.
Because they are polymorphic they are useful in linkage studies which attempt to locate
genes responsible for various genetic disorders.
Population Studies is done by looking at the variation of microsatellites in
populations, inferences are be made about population structures and differences, genetic
drift, genetic bottlenecks and even the date of a last common ancestor.

Forensics researchers use SSR loci, generally known in forensic applications as

Short Tandem Repeat loci, are widely used for forensic identification and relatedness
testing, and are a predominant genetic marker in this area of application. Another
application involves linking DNA samples with relatives of a missing person. Because
the lengths of microsatellites may vary from one person to the next, scientists have begun
to use them to identify criminals and to determine paternity by using DNA profiling or
"fingerprinting". The features that have made use of microsatellites attractive are due to
their relative ease of use, accuracy of typing, highly consistent and high levels of
polymorphism. The ability to employ PCR to amplify small samples is particularly
valuable in this setting, since in criminal casework only minute samples of DNA may be
available.

SSRs are used to detect sudden changes in population, effects of population

fragmentation and interaction of different populations. They are useful in identification
of new and incipient populations thus studied in conservation biology.The use of SSR
markers in breeding was first proposed by Tanksley (1983) and reviewed by Melchinger
et al. (1990). Marker assisted back crossing (MAB) is a vital tool in crop improvement
Now a days Back-crossed breeding in Maize (zea mays L.) has been extensively used to
transfer favourable alleles of monogenic traits from donor genotypes to elite inbred lines.
SSRs are used as molecular markers in breeding program as a diagnostic tool to trace the
presence of a target allele when direct selection is difficult or impossible.

Advantages:

 Relative ease of use

 High levels of accuracy
 High levels of polymorphism
 Very informative
 More alleles in one locus
 Shows high consistency in their polymorphism
 Abundant throughout the genome
 Avilability of large data through database
 Extensive research studies
 Availability of information about the flanking sequences
 Largely associated with non-repetitive DNA sequences(STS sites)
 Easy to scores
 REVIEW OF LITERATURE

The term optimization refers to adjustment of every component of the system to

bring the best possible results. Even a positive result can be optimized in terms of various
parameters like efficiency, cost, time etc. PCR has many qualitative characteristics such
as specificity, efficiency, sensitivity, fidelity. During experiment PCR can be failed for
many reasons in part due to the sensitivity of PCR to contamination causes amplification
of spurious of DNA fragments. So many procedures now a days being developed for
optimizing PCR condition. Primer design techniques are important in improving PCR
product yield and help full in avoiding the formation of unwanted products along with
use of different buffer components or polymerase enzymes helps to bridge over the
huddles of DNA amplification encountered during the reaction in process.

DNA fingerprinting using Maize SSRs is a very good in vitro technique to

develop DNA fingerprints to study polymorphism and other genetic studies in plant
including genome fingerprinting. These SSRs are abundantly present throughout the
plant genome, highly informative, and are easy to handle .More importantly from the
experiments. performed by many molecular biologists and scientists in the past ,like J.B.
Holland, S.J. Helland, N. Sharopova, and D.C. Rhyne in 1999.Y. Vigouroux, M.
McMullen, C. T. Hittinger, K. Houchins, L. Schulz, S. Kresovich, Y. Matsuoka, and J.
Doebley( 2003)Kejun Liu1, Major Goodman, Spencer Muse, J. Stephen Smith, Ed
Buckler, and John Doebley in 2001.B. Kalyan Chakravarthi1 and Rambabu Naravaneni,
Dept of Biotechnology, Sri Krishnadevaraya University, India.Genetics department,
Bhagawan Mahavir Medical Research Centre. Marko maras, Jelka Sustar-Vozlic, Branka
Javornik, Vladimir MeglicSS in 2006),it has been found that they show high level of
polymorphism as compared to other genetic markers.SSRs show best consistency in their
polymorphism which makes it a highly important marker,but for their amplification the
PCR has to be optimized as different primers to be used have different melting temp.
(Tm). SSR-based PCR makes the use of Touch-down PCR profile for DNA amplification
using simple sequence repeats.

SSRs are present in eukaryotic genomes (Hearne et al., 1992) and were described
in various plant species, such as soybeans (Akkaya et al., 1992), Arabidopsis thaliana
(Bell and Ecker, 1994), grapes (Browers et al., 1996), sugar beets (Morchen et al., 1996),
rice (Wu and Tanksley, 1993), and tropical trees (Condit and Hubbell, 1991).

From the experiments ( by Michele Morgante and M. Hanafey, Scotland) it was

found that Micro satellites are a ubiquitous class of simple repetitive DNA sequence,
thought to result from the mutational effects of replication slippage. Large-scale genomic
sequencing provided an opportunity to evaluate the abundance and relative distribution of
microsatellites between transcribed and non-transcribed regions. and the relationship of
these features to haploid genome size. Among species, the overall frequency of
microsatellites is inversely related to genome size and to the proportion of repetitive
DNA but remained constant in the transcribed portion of the genome. . The microsatellite
frequency is higher in transcribed regions, especially in the untranslated portions, than in
genomic DNA.
Simple sequence repeats or Microsatellites abound and are useful as molecular
markers in maize because they can be found in all chromosomes and contain a high-
polymorphism information content (Senior and Heun, 1993; Chin et al., 1996)Moreover,
they are inexpensive and very easy to handle since they require small amounts of
template DNA. Finally, information about primers that amplify SSRs aggregated from
Maize Genomic Database(MaizeGDB) and bought directly from specialized companies
e.g Qiagen. Despite these advantages, there are some practical difficulties. Microsatellite
alleles may differ by as few as one base pair, thus requiring samples to be run in high-
resolution agarose gels(Metaphor,USA). Most importantly, at first PCR conditions
needed to be optimized for each locus, since primers used in their amplification vary
both in size and nucleotide composition.

SSRS are preferentially associated with non-repeatative DNA in plant genome

described by .Michele Morgante, Michael Hanafey & Wayne Powell in 1996.Maize
(Zea mays L.) SSR loci are useful as genetic markers because they are numerous, occur
in every chromosome, and have a high content of polymorphism information.
Furthermore, they can be amplified by PCR (SSR-PCR) and the resulting fragments
resolved on agarose gels.

However, a major problem is that the primers used in the amplification process
often have different length and nucleic acid content, requiring optimization of PCR
amplification programs for each locus. So to circumvalent and get rid of such problems
Juliana Bernardi Ogliari, Raquel Boscariol and Senior etal in 1996 and Luis E.A.
Camargo developed "Touchdown" PCR programs where the annealing temperature (Tm)
is gradually decreased as the reaction takes place; however, this does not work well for
various loci and for this reason other variables, such as primer concentration, must be
considered. Optimal amplification conditions were successfully developed for a set of 7
SSRs and the selected SSRs used in this project are bnlg619, bnlg2238,
bnlg1191,bnlg1046, bnlg 244,dupssr28 and phi001.

The programs varied both in relation to the annealing temperature (Tm) and
primer concentration. Primers could be divided into four groups. A large group included
primers that amplified well in a basic previously published amplification program but
with different primer concentrations. A second group amplified in alternative programs
in which the annealing temperatures were changed so as to include the Tm of either both
primers or the highest Tm of the primer pair estimated based on their nucleotide
composition. A third group included primers that amplified in programs with Tm higher
than those estimated, and in a fourth group, with just two pairs of primers, the opposite
situation prevailed.

A two-stage touchdown amplification program was designed for each SSR based
on the Tm of both primers, calculated ( Newton and Graham etal, 1997 ) as from the
given formula Tm = [20C (A + T) + 40C (C + G) - 5oC] so that, when possible, both Tm
were included in the program. An initial denaturation step of 5 min at 95oC was used in
all programs, followed by a first stage consisting of 10 cycles. In this stage Tm , starting
from highest point was gradually decreased by 1oC following every 1 cycle with a
denaturation step of 95oC just after the initial denaturation. . This treatment was followed
by a second stage consisting of 30 amplification cycles, in which the lowest T m(55 oC)
remained constant. Denaturation and elongation steps were held constant for 1 min at
95oC and 1 min at 72oC, respectively, during second stage. An elongation step of 1 min at
72oC was performed in all programs at stage 2 followed by an elongation step of 72oC for
5 min for all profile. Annealing temperatures of primers was estimated on the basis of
Newton and Graham's (1997) formula does not not always correspond to the best Tm
observed, based on the signal strength of the amplicon. This was not unexpected, because
the authors had acknowledged that their formula was developed based on hybridization
experiments conducted at a higher salt concentration (1 M) and that the resulting Tm
might need adjustments for nucleotides longer than 20 bp, often the case in
microsatellites.
SSRs have acted as versatile tools and have found their own position in various
fields like taxonomy, physiology, embryology, genetic engineering, etc. They are no
longer looked upon as simple DNA fingerprinting markers in variability studies or as
mere forensic tools. Ever since their development, they are constantly being modified to
enhance their utility and to bring about automation in the process of genome analysis.
The discovery of PCR (polymerase chain reaction) was a landmark in this effort and
proved to be an unique process that brought about a new class of DNA profiling markers.
This facilitated the development of marker-based gene tags, map-based cloning of
agronomically important genes, variability studies, phylogenetic analysis, synteny
mapping, marker-assisted selection of desirable genotypes, etc. Thus giving new
dimensions to concerted efforts of breeding and marker-aided selection that can reduce
the time span of developing new and better varieties and will make the dream of super
varieties come true. These DNA markers offer several advantages over traditional
phenotypic markers, as they provide data that can be analysed objectively. In this article,
DNA markers developed during the last two decades of molecular biology research and
utilized for various applications in the area of plant genome analysis are reviewed.
Genetic fingerprinting in Maize is done by using an Agarose Gel System(Horizontal
electrophoresis).Amplified fragments were well resolved in 2.5% agarose gels containing
ethidium bromide at a concentration of 0.5 mg/ml of gel. Gels were run in 1X TBE TE
at 120 mAmp for minimum 2.5h.

DNA finger printing:

The term DNA-fingerprinting was introduced for the first time by Alec Jeffrey in
1985 to describe bar-code-like DNA fragment patterns generated by multilocus probes
after electrophoretic separation of genomic DNA fragments. The emerging patterns make
up an unique feature of the analysed organism and are currently considered to be the
ultimate tool for biological individualization and characterization. Recently, the terms
DNA fingerprinting/profiling is used to describe the combined use of several single locus
detection systems and are being used as versatile tools for investigating various aspects
of plant genomes. These include characterization of genetic variability, genome
fingerprinting, genome mapping, gene localization, analysis of genome evolution,
population genetics, taxonomy, plant breeding, and diagnostics. It is an in vitro technique
in which the banding patterns of DNA fragments from two or more different organisms
are compared.

“DNA fingerprinting also known as DNA typing or genetic fingerprinting, is a

method for identifying a organism by the particular structure of their DNA. It gained its
name because the structure of the DNA of each organism is different, and hence, just as
each of individual is unique with respect to the pattern of fingerprints, so they can be
identified from their DNA”.The DNA is run in a high resolution agarose gel to see the
banding pattern after electrophoresis and identified under Uv-transilluminator and Gel-
doc system.

SSR-PCR:

When compared to the general PCR this type of PCR uses a slightly modified profile
for the reaction.. This is so named as it uses SSR- loci of the genomic DNA having the target
sequence repeat motif to be amplified and the primers used here are complementary to the
flanking sequence of ssr repeats. Generally this uses Touch-down profiles for the desired
DNA amplification.

Touchdown PCR:-
Touchdown PCR(Senior, Chin, E.C.L etal,1996) profile is another modification
of conventional PCR that may result in a reduction of nonspecific amplification. The
main objective of this type of PCR is to make the different type of primers which are
used in the PCR to be compatible with the reaction condition. Ultimately making them
suitable for actively reacting in the PCR. It involves the use of an annealing temperature
that is higher than the target optimum in early PCR cycles. The annealing temperature is
decreased by 1°C every cycle or every second cycle until a specified or 'touchdown'
annealing temperature is reached. The touchdown temperature is then used for the
remaining number of cycles. This allows for the enrichment of the correct product over
any non-specific product.

Touchdown polymerase chain reaction or touchdown style polymerase chain

reaction is a method of polymerase chain reaction by which primers will avoid
amplifying nonspecific sequence. The temperature at which primers anneal during a
cycle of polymerase chain reaction determines the specificity of annealing. The melting
point of the primer sets the upper limit on annealing temperature. At temperatures just
below this point, only very specific base pairing between the primer and the template will
occur. At lower temperatures, the primers bind less specifically. Nonspecific primer
binding obscures polymerase chain reaction results, as the nonspecific sequences to
which primers anneal in early steps of amplification will "swamp out" any specific
sequences because of the exponential nature of polymerase amplification.

The earliest steps of a touchdown polymerase chain reaction cycle have high
annealing temperatures. The annealing temperature is decreased in increments for every
subsequent set of cycles (the number of individual cycles and increments of temperature
decrease is chosen by the experimenter). The primer anneal at the highest temperature
which is least-permissive of nonspecific binding that it is able to tolerate. Thus, the first
sequence amplified is the one between the regions of greatest primer specificity; it is
most likely that this is the sequence of interest. These fragments will be further amplified
during subsequent rounds at lower temperatures, and will out compete the nonspecific
sequences to which the primers may bind at those lower temperatures. If the primer
initially (during the higher-temperature phases) binds to the sequence of interest,
subsequent rounds of polymerase chain reaction can be performed upon the product to
further amplify those fragments.

To check whether the PCR generated the anticipated DNA fragment (also sometimes
referred to as amplimer), agarose gel electrophoresis(horizontal) was employed for size
separation of the PCR products. The size(s) of PCR products is thereby determined by
comparison with a DNA ladder,GeneI,Bangalore( λ DNA), which had DNA fragments of
known size(100bp), ran on the gel alongside the PCR product

Parameters of SSR-PCR:

1. Sensitivity and Specificity of increased by:

 Increasing concentration of primers (0.2-1 µM final concentration)

 Increasing concentration of Taq DNA polymerase (0.25 – 0.5 units)

 Increasing number of cycles.

 Increasing/ decreasing annealing temperature.

 Increasing/ decreasing annealing and extending time.

 Decreasing concentration of Taq DNA polymerase.

 Reducing annealing time.

 Reducing extending time.

 Increasing annealing temperature.

 Decreasing number of cycles.

 Using Hot Start technique.

2. Efficiency for the yield of PCR products are improved by increasing:

 dNTP concentration (0.1-0.5 mM final concentration)

 Mg2+ concentration (1-5 mM final concentration)

 Taq DNA polymerase concentration.

 Annealing time, Extending time and Number of cycles.

3. Efficiency & Fidelity of PCR increased by :

 Decreasing dNTP concentration

 Decreasing Taq DNA polymerase conct.

 Decreasing Mg2+ conc.

 Minimizing reaction time at high temp.

 Decreasing number of cycles.

 MATERIALS AND METHODS

Materials:
Chemicals used:

 Liquid nitrogen

 Crushed ice

 CTAB buffer

Extraction buffer.


 ß-mercaptoethanol

 Chloroform:isoamyl alcohol (24:1)

Ice-cold iso-propanol


 70% ethanol

 Tris-EDTA (TE) buffer

 TBE buffer (Tris borate EDTA)

Rnase Solution


 3.0 M sodium acetate

 Absolute ethanol
  Agarose(low resolution &metaphor high resolution agarose powder)

 Gel loading dye

 Lambda DNA standards

10 mg/ml Ethidium bromide (EtBr) staining solution,



Instruments and glass wares used:

 Mortar and pestle.

 Homogenizer.

Sterile Spatula 2.0 and 1.5 ml microcentrifuge tubes



 Sterile pipet tips (1000ul, 200ul, 10ul)

 Eppendorf tubes(1.5 ml)

Micropipets (100ul, 200ul, 10ul)



Water bath at 650C and 370C



 Microcentrifuge.

Thermal cycler machine(BIO-RAD,USA)



Horizontal Electrophoresis Apparatus



 UV Transilluminator.

 Gel-doc system

Other miscellaneous:

Nitrile gloves,deep freeze..Etc

Plant Materials used for the project:

 For DNA sample isolation, leaves of five different Maize plants were

collected from Rajendra Nagar Plant Nursery,Hyderabad

Procedure for DNA isolation:

DNA isolation involves at least 3 essential steps , cell-breakage, removal of
protein and RNA, and DNA precipitation by which DNA is separated from all cell
components such as RNA, protein, lipids, polysaccharides, nucleotides, amino acids, ions
and water. The quality and amount of purified DNA is then assessed by Agarose
electrophoresis or spectrophotometry.Fowlling steps involved in the genomic DNA
isolation which are given below.

100 mg of fresh 3rd/4th leaf was crushed in an eppendorf tube containing 500 µl


of 2X CTAB buffer (Extraction buffer). by homogenizer.

The extraction buffer consisted of 2 %( w/v) CTAB (Cetyle trimethyle



ammonium bromide), 1.4M NaCl, 20mM EDTA (pH 8.0), 100mM Tris – HCl

(pH 8.0) and 2% (V/v) β - Mercaptoehtanol

 Ground tissue was incubated at 650C for 1Hr.

 Ground tissue was cooled to room temperature.
 500 µl Chloroform: Isoamyl alcohol (24:1) solution was added and vortexed
vigourously.
 The eppendorf tube was centrifuged (Sigma-2000) at 14000 RPM for 5 min at
room temperature
 The supernatant is added to a new eppendorf tube containing 400 µl isoproponol
 The tube was inverted for 50 times and centrifuged at 14000 RPM for 5 Min at
room temperature
 Supernatant was discarded and the DNA pellet is washed with 500 µl 70%
ethanol
 DNA pellet was air dried in laminar air-flow (Syngene) for 5 Min at room
temperature after pouring off the ethanol.
  DNA pellet was resuspended in 1X T10E1(Tris-Cl 10mM,EDTA 1mM) buffer pH

Name of Sample O.D at 260nm for O.D at 280nm O.D Ratio

1µL/150µL for 1µL/150µL
Z. mays 0.074 0.039 1.90
Z. hirta 0.073 0.033 2.21
Z. rostrata 0.138 0.072 1.92
Z. marcosperma 0.054 0.032 1.69
0.059 0.032 1.84
8.0
 .Resuspended DNA was incubated at 650C for 1Hr to complete DNA rehydration.
 DNA was stored at -200C(Tris-Cl 10mM,EDTA 1mM)

Procedure for DNA purification:

 The dissolved DNA was crude DNA and required further purification.

 The RNA was removed by giving RNase treatment.(10mg/ml)

 For 1 ml of DNA solution , 2μl of RNase was added and solution was
incubated in a water bath at 37˚C for 1hr

 200 μl of phenol was added, and then shake for 10 mins. The solution was then
centrifuged at 10,000rpm for 20 min. The supernatant was then collected.

 75 μl volume of Chloroform: Isoamyl alcohol (24:1) was added and gently

mixed for 10 minutes

 The solution was then centrifuged at 10,000rpm for 20 min.

Table -1 showing the quality of DNA

MOLECULAR CHARACTERIZATION:
Checking the DNA Quality and Quantity:
Agarose electrophoresis provides a rapid and convenient way to measure the Quantity of
DNA and visualize its quality at the same time.
 At first Gel mold was prepared according to manufacturer’s instructions.
 0.8 % Agarose gel was prepared. For example, to make a 50 ml gel, 0.4 g of
agarose was weighed, place in a dry 500-ml Erlenmeyer flask, and add 50 ml of
0.5x TBE buffer. Microwave for at least 3 min or until the agarose is evenly
melted. The solution cooled to about 500C or until able to hold the flask on the
palm of hand. Pour the gel into the mold and allow the gel to polymerize for
about 1hour.
 The electropheresis tank was filled with enough 0.5x TBE buffer to cover the
whole gel.
 Carefully the wedges were removed also comb/s and the gel was placed along
with the mold in the tank.
 Using a 10-µl micropipet, 3.0 µl DNA solution was mixed with about 1 µl of gel
loading dye on a parafilm or microplate. Mix all the samples with dye.
 To be able to estimate the DNA concentration of the samples, DNA
standards were prepared by mixing 10 ng/ml, 20 ng/ml and 100 ng/ml lambda
DNA with dye as above.
 Carefully the samples were loaded into each well. Load the lambda DNA
standards on the first three lanes and the rest of the samples on the following
wells.
 Electrophoresis was allowed to run at 100 volts, for about 1-2 hours or until the
fast blue dye (Bromphenol blue) reaches the end of the gel.
 The gel was stained in Ethidium bromide solution.
 The gels was placed on a UV transilluminator and capture the gel image with a
camera.
 The lanes were identified and estimated DNA concentration was estimated by
comparing the DNA samples with the lambda DNA standards.

Master Mix preparation :

Calculations:

1.Buffer (TBE) :
Initial concentration M1=10x
Initial volume V1= ?
Final Concentration M2=1x
Final Volume V2=25µl
M1V1=M2V2
V1=M2V2/M1
= 1*25µL/10µL
=2.5 µL
2.MgCl2:
Initial concentration M1=10x
Initial volume V1= ?
Final Concentration M2=1x
Final Volume V2=25µl
M1V1=M2V2
V1= 1.5 µM*25 µL/25 µM
=1.5 µL
3.dNTPs:
Initial concentration M1=10x
Initial volume V1= ?
Final Concentration M2=1x
Final Volume V2=25µl
M1V1=M2V2
V1=200 µM*25 µL/2.5mM
=2.0 µL
4.Taq. Polymerase:
Stock concentration = 5 unit per 1 µL
So 0.5 unit will consume 0.10 µL in the master mix.
5.Primers:
Initial concentration M1=10x
Initial volume V1= ?
Final Concentration M2=1x
Final Volume V2=25µl
M1V1=M2V2
V1= 0.4 µM*25 µL/5 µM
=2 µL
6.Template DNA=1 µL
Table-2 Showing the components of PCR with their respective conc

Components Stock conc. Final conc. Final volume

For 1 tube For 7 tubes
DNA 85ng/µL 30 µL 1 µL 7 µL

Buffer 10x 1x 2.5 µL 17.5 µL

dNTPs 2.5mM 1.5 µM 2.0 µL 14.0 µL

MgCl2 25 µM 1.5 µM 1.5 µL 10.5 µL

Taq poly. 5 Unit/ µL 0.5 Unit 0.10 µL 0.70 µL

Primer-1(F) 5µM 0.4 µM 2.0 µL 14.0 µL

Primrer-2(R) 5µM 0.4 µM 2.0 µL 14.0 µL

Water 13.9 µL 97.30 µL

Total 25 µL 175 µL

147µl of master mix was distributed into seven tubes as follows:

21µl of master mix was put into each of the seven tubes
+
02µl of each primer sets (forward & reverse) inserted into each tube
Final volume in each tube= 21+02+02 (template DNA + for. Primer + rev. primer)
= 25µl .Total reaction volume 25.0 µL

Calculation of Tm (melting temp.)of 7 primer sets used

in the experiments:
An updated list of maize SSR markers were accessed from the MaizeGDB
Database while primers used were from Qiagen company(USA) .There are many
formulas proposed by many research workers and biochemist out those the formula used
in our experiment which is based on the Guanine and cytosine content of the primer
used. The formula(Newton and Graham, 1997) is given below.
Tm of the primers = 64 0C+410C *(No of G’s + No of C’s present in the given primer)/N
Where N =length of the given primer(no of oligonucleotides present).This formula is
applied for those primers whose length is >14.
G=guanine
C=cytosine

EXPERIMENTS:

Profile optimization(OPTIMIZATION OF PCR AMPLIFICATION OF MAIZE SSR

LOCI TO DEVELOPE DNA FINGERPRINT):

Optimization of PCR profile was performed to ensure the good and substantial
amplification of DNA,to be used for genetic fingerprinting and scoring polymorphism in
SSRs.For this a minimum of five trails was done whose profiles are described below.(
see also fig no.6a- 6e)

[PCR Trail-1]
This trail was done to scrutinize whether the annealing temp. (55 0C) of step-2 in cycle-3
is suitable for DNA amplification for thermal cycler profile.(touchdown )
1. Initial denaturation 950C for 5 m
2. Denaturation 950C for 1 min
3. Annealing 650C for 1 m in touch down process of 1degree decrease in temp.in
each subsequent cycle.
4. Extension 720C for 1 m
5. Cycle goes to step 2, for 10 times
6. Here another cycle starts as follows
7. Denaturation 950C for 00.45 sec
8. Annealing 550C for 45 sec
9. Extension 720C for 45 sec
10. Cycle go to step 5 for 20 times
11. Final Extension 720C for 5 m
12. Soak at 40C
Fig-6a showing PCR Profile for Trail-1

[PCR Trail-2]
This trail was done to check whether the modification in no of repeats in cycle-3 and
annealing time is suitable for desired DNA amplification or not.
1. Initial denaturation 950C for 5 m
2. Denaturation 950C for 1 min
3. Annealing 650C for 1 m in touch down process of 1degree decrease in temp.in
each subsequent cycle.
4. Extension 720C for 1 m
5. Cycle goes to step 2, for 10 times
6. Here another cycle starts as follows
7. Denaturation 950C for 00.45 min
8. Annealing 550C for 1 m
9. Extension 720C for 45 sec
10. Cycle go to step 5 for 30 times
11. Final Extension 720C for 5 m
12. Soak at 40C.
Fig-6b showing PCR Profile for Trail-2

[PCR Trail-3]
This trail was done to ensure whether the annealing temp of 530C is suitable for desired
DNA amplification or not.
1. Initial denaturation 950C for 5 m
2. Denaturation 950C for 1 min
3. Annealing 650C for 1 m in touch down process of 1degree decrease in temp.in
each subsequent cycle.
4. Extension 720C for 1 m
5. Cycle go to step 2, for 10 times
6. Here another cycle starts as follows
7. Denaturation 950C for 00.45 sec
8. Annealing 540C for 1 m
9. Extension 720C for 00.45 m
10. Cycling go to step 5 for 30 times
11. Final Extension 720C for 5 m
12. Soak at 40C.
Fig-6c showing PCR Profile for Trail-3

[PCR Trail-4]
This trail was done to check whether all the components and modification in annealing
temp of 540C is compatible for the desired DNA amplification or not.
1. Initial denaturation 950C for 5 m
2. Denaturation 950C for 1 min
3. Annealing 650C for 1 m in touch down process of 1degree decrease in temp.in
each subsequent cycle.
4. Extension 720C for 1 m
5. Cycle go to step 2, for 10 times
6. Here another cycle starts as follows
7. Denaturation 950C for 00.45 min
8. Annealing 540C for 1 m
9. Extension 720C for 00.45 m
10. Cycling goes to step 5 for 30 times
11. Final Extension 720C for 5 m
12. Soak at 40C for indefinite time.
Fig-6d showing PCR Profile for Trail-4

[PCR Trail-5]
This trail was to check whether the annealing temp of 550C along with extension time
of 01.00 min for cycle-3 is suitable for desired DNA amplification or not.

1. Initial denaturation 950C for 5 m

2. Denaturation 950C for 1 min
3. Annealing 650C for 1 m in touch down process of 1degree decrease in temp.in
each subsequent cycle.
4. Extension 720C for 1 m
5. Cycling go to step 2, for 10 times
6. Here another cycle starts as follows
7. Denaturation 950C for 00.45 min
8. Annealing 550C for 1 m
9. Extension 720C for 1 m
10. Cycle goes to step 5 for 30 times
11. Final Extension 720C for 5 m
12. Soak at 40C for indefinite time.
Fig-6e showing PCR Profile for Trail-5

Agarose gel-electrophoresis for amplified products of PCR trails:

 1.0% agarose solution was prepared by mixing 0.50 gm.of agarose in 50ml TBE.
If a small DNA molecule is analyzed,
 The solution boiled to dissolve the agarose, preferable in a microwave oven.
 The solution cooled down to about 500C at room temperature, or water bath.
 2µL ethidium bromide was added to stock per 50ml gel solution...
 The gel was put into a tank with 0.5x TBE. Ethidium bromide at the same
concentration was added to the buffer.
 10µl of template DNA(Maize leaves of five plants at a nursery, Rajendranagar,
Hyderabad ) was loaded into each well and gel was run at a power supply of 120
mAmp(constant current) for 1.30 hrs.
 Power supply=120 mAmp(constant current). Time of run=1.15 hrs

Scoring polymorphism in SSRs:

A series of seven PCR setups were performed in this project to study
polymorphism in SSRs, in the selected seven loci of Maize plants one after another.The
profile choosen for all these PCR setups was like this:
  Initial denaturation 950C for 5 m
 Denaturation 950C for 1 min
 Annealing 650C for 1 m in touch down process of 1degree decrease in temp.in
each subsequent cycle.
 Extension 720C for 1 m
 Cycling go to step 2, for 10 times
 Here another cycle starts as follows
 Denaturation 950C for 00.45 min
 Annealing 550C for 1 m
 Extension 720C for 1 m
 Cycle goes to step 5 for 30 times
 Final Extension 720C for 5 m
 Soak at 40C for indefinite time.
Table-3showing the name of SSR and their corresponding primer sequences.

S.L PCR Name of Forward Primer Reverse Primer

. Setup SSR
No. No. used
1 1 Bnlg244 5’GATGCTACTACT 5’CTCCTCCACTCA
GGTCTAGTCCAGA3’ TCAGCCTTGA3’
2 2 Bnlg2238 5’ TGCCACTCAAG 5’TTCTGATTGCAGT
CCTTCTTTT3’ GCAGACCCTCCTCCACT3
’
3 3 Bnlg619 5’ACCCATCCCACT 5’GCTTTCAGCGAAT
TTCCACCTCCTCCT3’ ACTGAATAACGCGGA3
4 4 Bnlg1191 5’AATCATGCGTAG 5’GCCAGAGGAAAAAG
GCGTAGCT3’ AAGGCT3’
5 5 Bnlg1046 5’TGAGCCGAAGCT 5’GATGCAAAGGAGG
AACCTCTC3’ TTCAGAA3’
6 6 Dupssr28 5’GAAGGAAGCCTT 5’CTGGAGTGCTGGTCT
TGTTACAAGT3’ TGTTAT3’
7 7 Phi001 5’GATGCTACTACTG 5’CTCCTCCACTCATCAG
GTCTAGTCCAGA3’ CCTTGA3’

Gel-electrophoresis for amplified products of PCR setups in polymorphism

study:
 Here 2.5% high resolution Agarose gel(40 ml Tbe buffer )is used for
electrophoresis
Buffer used=TBE(1X)
Power supply=120 mAmp(constant current).
Time period of run=2.5 hrs.

DEVELOPMENT OF FINAL DNA FINGERPRINT:

Finally fingerprints of DNA was developed by taking all primers in one PCR
reaction. Here DNA from Zea mays L. was taken as template.

Gel-electrophoresis for amplified products of PCR setups in polymorphism study:

Here 2.5% high resolution Agarose gel(40 ml TBE buffer )is used for
electrophoresis
Buffer used=TBE(1X)
Power supply=120 mAmp(constant current).
Time period of run=2.5 hrs.
Agarose used is High resolution Metaphor agarose of Sea kem Lonza, USA

RESULTS
 This project work was aimed at developing DNA fingerprints from five species
of Zea through SSR-PCR using SSR as a molecular marker. (Touch-down profile)

Genomic DNA isolation and quantification:

For isolation of DNA, the CTAB method (Doyle and Doyle, 1990) was adopted
for five Maize species which was slightly modified (Z.mays, Z.hiria, Z.curagua,
Z.marcosperma and Z.rostrata). Good quality of DNA was obtained as revealed by
agarose gel electrophoresis (Fig. 5) The concentration of DNA varied from 50ng/µl -
85ng/µL This was also checked in spectrophotometer. Finally it was diluted to 30ng/µL.

Primer selection:
The DNA amplification was tested by using 7 primers (Qiagen,USA) of GTMSZ
series namely i.e GTMSZ11, GTMSZ12, GTMSZ13, GTMSZ14, GTMSZ15, GTMSZ 16
and GTMSZ17. respectively.Primers were complementary to flanking sequence of a
particular SSR repeat and the names of such SSR are bnlg244, bnlg2238, bnlg619,
bnlg1191 , bnlg 1046 dupssr28 and phi001 .They were commercially pre-designed ones
and information about these being retrieved from Maize Genomic Database. These
primers are loci specific. PCR reaction using such primers was done in Touch-down
fashion. Fragment sizes of these primers were in the range of 380-120 bps.
PCR Profile optimization:
In PCR trail-1 using the described profile it was interpreted that out of 7 primer
sets only the primers like GTMSZ-16 and 17 (in 6 and 7 Fig no-7) showed good
amplification. Moreover primers13and15 (lane-3 and 5) showed less amplification. There
was no amplification in lane-2 and very less amplification in lane-5 and 3.So this
revealed that profile for this trail is not suitable for most of the primers. As most of the
primers produced very faint to faint bands. Thus modification was done in the profile by
choosing the increase in number of cycles in the PCR.( out of many parameters) .Now
the number of cycles are increased to 30 from 20 previously for next trail(stage-2)
In PCR trail-2,a good amplification was seen for few primers and few showed
faint bands of amplification also the primers in 6th and 7th well(Fig no-7) not showed
considerable amplification. There were very faint bands for primers like 12 and 15, an
indication of less amplification. Also there was no amplification for GTMSZ-16.So the
assumption is that there is any thing less in master mix or the annealing temp in stage-2.
is higher for for PCR.Thus modification was made at annealing temp of 53 0C in cycle3
(stage-2), for the next trail PCR trail-3 revealed that the profile setup for this trail was
only suitable for primers like GTMSZ-12,14 and17 respectively in lane-3,6,8(Fig no-
7).The temp of 530C is not suitable for maximum no of primers for DNA amplification.
So looking at the above results modification was made in this profile. Annealing temp.
of PCR in cycle-3 modified to 540C for next trail.PCR trail-4 showed that, profile in this
PCR reaction was only favorable for primer GTMSZ-17 in well-8(Fig no-7).Very faint to
faint bands almost all the primers scored.
The amplification results showed that annealing temp 0f 54 0C is not suitable
when compared with the previous trail(trail-3) As annealing temp of 550C(in trail-2)
showed very good results when compared to both temperatures of 530C and 540C.This
temp is finally selected for amplification but to get more better results extension time in
cycle-3 (stage-2) is modified to 1.00 min for next trail. Here modification was aimed to
get better amplification for primer-16 PCR trail-5 showed that all the seven primers
shows good and well recognizable amplification along with well resolved (Figno-7)
distinct DNA bands. As a result of which the thermal cycler profile in trail -5 was
considered as the best condition for PCR among all five trails .So trail-5 profile recruited
for the development of final fingerprint and experiments to study polymorphism in SSR.
Information about the size of amplicons in these five trails given below in table-4

Table-4 showing the Size of the Amplicons in each lane where the no indicates the size in bps.

Name of Species Lane1 Lane2 Lane3 Lane4 Lane5 Lane6 Lane7

Z.mays 152 183 223 245 188 200,234 122,202

Z.hirta 152 200 223,264 245 202 200,234 122,202

Z.rostrata 144 172 223,249 200,262 171 190 122,202

Z.marcosperm 152 183 249,273 200,262 188 190,224 122,202

a
Z.curagua 153 178,212 235 195 188 190 122,202

Simle sequence repeats (SSR) Analysis:

Seven pre-established and pre-designed locus specific polynucleotide primers from
GTMSZ series were tested using 5 (Rajendra nagar,hyderabad) accession and out of them
5 were showing distinct and well resolved polymorphism for the given specified SSR(in
their banding pattern).Other two were monomorphic . A total of 21 bands were resulted
from the 5 primers from GTMSZ11-15.All the 5 species produced distinct reproducible
amplicons. (fig.no.8 a&b) . A total of 14 bands amplified were all polymorphic.The
product sizes of SSR amplification is depicted in table -5
Table -5 Showing the size of the Amplicons prodused during polymorphism study in each lane
of gel.

Scoring polymorphism in SSR:-

Name of Lane -1 Lane-2 Lane-3 Lane-4 Lane-5

Primer
GTMSZ11 149bps 150bps 142bps 150bps 151bps
GTMSZ12 185bps 196bps 172bps 181bps 174&214bps
GTMSZ13 218bps 219&280bps 212&250bps 248&281bps 232bps
GTMSZ14 226bps 232bps 201&266bps 202&215bps 196bps
GTMSZ15 185bps 212bps 169bps 187bps 183bps
GTMSZ16 198bps 220bps 190bps 192bps 198bps
GTMSZ17 118&214bps 118&216bps 117&214bps 119&215bps 117&216bps
From PCR setup-1 it was quite clear that the ssr in locus bnlg244 is
distinctly polymorphic. The SSR in this locus showed single polymorphic and unique
band.There was no monomorphic band. PCR setup -2 revealed that the SSR in locus
bnlg2238 showed 4 polymorphic band along with 3 monomorphic band.more over no
monomorphic bands for this SSR.From PCR setup -3 it was interpreted that the SSr in
locus bnlg619 with having single monomorphic,polymorphic and unique band
respectively.From experiment-4 it was cleared that the SSR in locus bnlg1191 has two
polymorphic and unique bands. There were no monomorphic band at all.
From PCR setup -5 it was concluded that there was only one polymorphic and
unique band for this SSr for the locus bnlg1046. monomorphic band were not sited here.
PCR setup 6 & 7 was used for the loci dupssr28 and phi001, did not show any
polymorphism. How ever the ssr which is present in the locus dupssr28 showed one
unique band and the ssr in phi001 showed 2 monomorphic band,but no unique band.
From the above results one can establish that out of 7 SSR loci the SSRs present in 5 loci
namely bnlg244, bnlg2238, bnlg619, bnlg1191 & bnlg 1046 are distinctly
polymorphic.The loci dupssr28and phi001 are monomorphic.
Banding pattern and Amplification:
Table-6 showing the banding pattern of SSR
 Sl Primer name forw
no

Seven primers i.e GTMSZ-11 to GTMSZ-17 produced 21 bands, and of which 10

1 GTMSZ11
were polymorphic and only four were monomorphic. The amplicons were observed in
the range of >380 to 130 base pair. The details of SSR banding pattern and amplification
5’GA
with different primers and plant species are presented in Table-6.
The result of final fingerprint showed that all the seven primers were compatible
CTG
enough to give sufficient amplification with the help of above described (PCR trail-5)
touch down PCR profile. The Fig no-9 revealed that all the seven primers showing
AGA
2 GTMSZ1 2
ample DNA amplification starting from LANE1-LANE 7.The size of the amplicons
presented in Table-7 given below.
5’TG
Table-7 showing the size of the amplicons in final fingerprint
GCC
LANE NO 1 2 3 4 5 6 7
SIZE OF 150 196 220&282 231 213 220 118&216
AMPLICONS
IN bps 3 GTMSZ13 5’AC
CTT
TCC

4 GTMSZ14 5’AA
 DISCUSSION

From our results and above known literature in this project, particularly the trails
made for optimizing the PCR (from trail 1-5) conditions, we showed that the thermal
cycler profile having annealing temp of 550C in cycle -3 (Stage-2) and along with an
extension time of 1 min. (Step-3,stage-2) shows a very good result of DNA amplification
in these experiments. Here touchdown-PCR(Senior etal,1996) was used which is is a
great advantage over the conventional PCR as it is helpful in synchronizing all the
different primer sets used in one PCR reaction simultaneously. Using this PCR all the
primers are used simultaneously in one reaction tube and making them efficient for
achieving a distinct amplification but still having different Tm for different primers.In
trail 2 and 5 it was found that the above described profiles for PCR are the most suitable
conditions among all the experiments.The PCR setups from 1 to 7 which were performed
for establishing polymorphism in SSR revealed that , SSR used in the experiments was
showing a convincing and recognizable polymorphism in all the DNA samples for five
loci (Five different Maize leaf DNA sample from five different maize plants). All the 7
primers used in the project having complimentarily to the flanking region of the SSR at
the 3’ end of template DNA successfully showed amplification and they were helped in
all the experiments for establishing polymorphism.

These SSRs used in the experiments shows polymorphism in the Maize plant .
SSRs such as bnlg244,bnlg228,bnlg619,bnlg1191 in five loci out of 7 loci shows
polymorphism So the banding pattern of amplified DNA was different in all DNA
samples which were tested for all the 7 primer sets used during the polymorphism
experiments .So, one can say SSRs present in these loci can be used as an polymorphic
molecular marker.
 CONCLUSION
Crop improvement programs relies on the ideas and information about genetic
diversity present within the germplasm.Genetic characterization using phenotypic
characters is a traditional method. However more versatile and sophisticated molecular
marker techniques have replaced conventional techniques for characterizing genetic
diversity which is determined by looking at the banding patterns of DNA run on a gel.
The SSR based PCR using primers which flanks the simple sequence repeats provides a
good and handy laboratory technique for study of DNA amplification and genetic
fingerprinting.More importantly in this type of PCR Touch-down thermal cycle profile is
used for different primer sets having different annealing temperature is a huge boost over
the traditional method. This type of touch-down thermal cycle helps in optimizing all the
condition of the effective activity of all the primers and making them compatible for the
PCR reaction. That’s why the primers efficiently performs in a single protocol of thermal
cycler profile. Hence microsatellite based PCR is a very good technique of developing
DNA finger prints of different plant DNA which has got many applications in the field of
molecular biology and plant breeding.
The SSRs used in such PCR are highly polymorphic, co-dominant, abundant
thorough out the genome. So they are very helpful in studying the agronomic traits
present in maize plant and ultimately selecting these traits for the plant breeding
program. The marker based plant selection helps the plant breeders to establish the highly
productivity traits and to study genetic distance between different plants for molecular
biologists. SSRs based molecular markers are very highly informative, easy to handle
and shows a consistent result of polymorphism through DNA finger printing and the
protocols for SSRs based PCR is not highly sensitive.
Hence these SSRs are having great advantage over other DNA markers. So it is
concluded that PCR using the microsatellite (SSRs) is an excellent tool for the study of
DNA finger printing and DNA amplification using Touch-down PCR profile.DNA finger
printing using SSRs as the target sequences helps in developing DNA fingerprints.
 SUMMARY
 Polymerase chain reaction using SSRs provides an excellent molecular
tool for developing DNA fingerprints and studying polymorphism also.
This type of SSR based PCR uses touchdown PCR profile to make all the
primer sets compatible in a single PCR reaction which is having different
melting temperature.

 Touchdown PCR has got a great advantage over conventional PCR as it

reduces the time consumption for different PCR primers

 Moreover for DNA finger printing the PCR has to be optimized accurately
so that all the primers would perform efficiently to give the desired the
amplification by acting simultaneously in one directions. This helps in
development of DNA finger print with effective and consistent results

 The SSRs which are used in such experiments are very helpful to study
the genetic distance between plants. These SSR molecular markers highly
polymorphic, highly informative, co-dominantly inherited and abundant
through out the Maize plant

 Alsowhich, SSRs show good consistency in their results. So this

molecular marker has got many advantages over other genetic markers for
the study of genome analysis, especially for the plant breeders.
 BIBLIOGRAPHY

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
Wu, K.S. and Tanksley, S.D. (1993). Abundance, polymorphism and
genetic mapping of microsatellites in.rice. Mol. Gen. Genet

 9.
 1Departamento de Fitotecnia, Centro de Ciências Agrárias, Universidade Federal
de Santa Catarina, Caixa Postal 476, 88034-001 Florianópolis, SC, Brasil. Fax:
+55-48-334-2014.
2
Bursar RHAE/CNPq.
3
Departamento de Entomologia, Fitopatologia e Zoologia Agrícola, ESALQ/USP,
Caixa Postal 9, 13418-900 Piracicaba, SP, Brasil. Send correspondence to
L.E.A.C. Fax: +55-19-434-4839; E-mail: leacamar@carpa.ciagri.usp.br
 ). | Article | PubMed | ISI | ChemPort
 [ Links ]
 14 J
 State University, Raleigh, NC 27695, U.S.A.”
 . Department of Agronomy, University of Missouri—Columbia, Columbia, MO 65211, U.S.

 review of liter datas

  15.”
 16. 3 Remington, D. L., Thornsberry, J. M., Matsuoka, Y.,
Wilson, L. M., Whitt, S. R., Doebley

 17.J., Kresovich, S., Goodman, M. M. & Buckler, E. S.

(2001) Proc. Natl. Acad. Sci. USA 98,
 11479–
 18. ‘’Genetic Structure and Diversity among Maize Inbred Lines as
Inferred from DNA
 Microsatellites “”Department of Statistics, North Carolina State
University, Raleigh, NC 27695,
 2Department of Crop Science, North Carolina State University, Raleigh, NC
27695,
 3Crop Genetics Research and Development, DuPont Agriculture and Nutrition,
Pioneer Hi-Bred
 International, Johnston, IA 50131,
 4USDA-ARS and Department of Genetics, North Carolina State University,
Raleigh, NC 27695,
 5Laboratory of Genetics, University of Wisconsin, Madison, WI 53706

 19. “
SSR marker based DNA fingerprinting and diversity
 study in rice (Oryza sativa. L)”
 B. Kalyan Chakravarthi1 and Rambabu Naravaneni2
 20. “The efficiency of SSR markers in genetic
 diversity estimation and gene pool classification of
 common bean (Phaseolus vulgaris L.)” Marko MARAS1, Jelka
ŠUŠTAR-VOZLIČ2, Branka JAVORNIK3, Vladimir M

Abstract: Sequence databases could be efficiently exploited for

development of DNA markers if it were known which
gene regions reveal the most polymorphism when amplified by PCR. We
used PCR primer pairs that target specific
regions of previously sequenced genes from Zea species. Primers were
targeted to amplify 40 introns,
24 exons, and 23 promoter regions within 54 maize genes. We surveyed 48
maize inbred lines (previously assayed for
simple-sequence repeat (SSR) polymorphism) for amplification-product
polymorphism. We also developed primers to
target 14 SSRs and 12 introns within 18 Avena genes, and surveyed 22
hexaploid oat cultivars and 2 diploid Avena
species for amplification-product polymorphism. In maize, 67% of promoter
markers, 58% of intron markers, and 13%
of exon markers exhibited amplification-product polymorphisms. Among
polymorphic primer pairs in maize, genotype
diversity was highest for SSR markers (0.60) followed by intron markers
(0.46), exon markers (0.42), and promoter
markers (0.28). Among all Avena genotypes, 64% of SSR markers and 58%
of intron markers revealed polymorphisms,
but among the cultivars only, 21% of SSR markers and 50% of intron
markers were polymorphic. Polymorphicsequence-

1. tagged sites for plant-breeding applications can be created easily

by targeting noncoding gene regions

Introduction
Public databases of plant gene and protein sequences represent
a valuable and growing resource for plant genetics
and breeding. For example, simple-sequence repeats (SSRs)
can often be detected in gene sequences simply by searching
for diagnostic repeat sequences. Each SSR is a tandem repeat
of one or more short, simple sequences of two to six
nucleotides. Polymorphism is detected using oligonucleotide

1. primers complementary to conserved sequences flanking re

1066 Genome Vol. 44, 2001

peats in a polymerase chain reaction (PCR). Gel electrophoresis
can then be used to separate PCR amplification
products according to size, allowing detection of differences
in the number of repeats targeted and amplified. SSRs are
favored for plant-breeding and genetics applications because
they are abundant in plant genomes, highly polymorphic
within species, relatively rapid and inexpensive to assay, and
can be used to identify specific chromosomal regions consistently
across populations (Chen et al. 1997; Chin et al. 1996;
Senior et al. 1998; Smith et al. 1997; Taramino and Tingey
1996). SSR development in oat (Avena sativa L.) lags behind
that in other major cereal crops, with only 16 SSRs exhibiting
polymorphism between cultivated genotypes
reported to date (Li et al. 2000).
SSRs can be identified empirically by screening DNA
libraries for repeat motifs via hybridization and sequencing
candidate clones (Li et al. 2000; Taramino and Tingey
1996), applying SSR primers from related species (Li et al.
2000; Westman and Kresovich 1998) or mining sequence
databases (Chin et al. 1996; Senior and Heun 1993; Wang et
al. 1994). GenBank, a database supported by the National Center
for Biotechnology Information (http://www.ncbi.nlm.nih.gov/),
is the major public source of plant gene sequence information.
As of July 2001, GenBank contained 111 319 total sequences
and 214 intron-containing sequences from Zea
mays L. and 186 854 total sequences and 1020 introncontaining
sequences from Arabidopsis thaliana. Resources
for Avena species, however, are significantly smaller. The
database included 767 cDNA, genomic, chloroplast, intron,
spacer, and repeat sequences from 10 species in the genus
Avena. A majority of the sequences are mRNA or cDNA sequences
from A. sativa, and these 644 sequences can be
searched for SSRs. GenBank contains only 12 Avena
genomic sequences containing introns, 2 of which are from
chloroplasts. Because of the limited availability of sequence
information from some crop species, such as oat, we want to
develop methods to maximally exploit the available sequence
data for DNA marker development.
One approach to exploiting limited sequence information
is to develop PCR primers targeting specific gene regions to
determine which regions provide sufficient amplification
product length variation for use as DNA markers. Genomic
DNA sequences (in contrast to cDNA sequences) from the
public databases often indicate the positions of exons,
introns, and promoter regions within the primary gene sequence.
Therefore, genomic-sequence information can be
used to develop PCR primers flanking the exon, intron, or
promoter regions of known genes with high specificity. It
may be possible to use such PCR primers as an alternative
source of DNA markers that share the advantages of SSRs in
variability, specificity, speed, and inexpensiveness.
DNA sequences that do not code for protein products are
potentially more variable among alleles within a species because
the fitness consequences of sequence variations in
those regions are expected to be smaller than variations
within coding sequences. Intron sequences evolve more rapidly
than exon sequences in both plants (Small and Wendel
2000) and mammals (Hughes and Yeager 1997). Under the
assumption that intron sequences evolve independently of
function, they have been used as estimates of “genetic time”
in phylogenetic studies (He and Haymer 1997; Johnson and
Soltis 1994). Further, length variation among intron alleles
within plant species has been reported. Chetelat et al. (1995)
developed an allele-specific PCR marker for the sucr gene
in tomato (Lycopersicon sp.) by designing primers to
amplify several exons and introns, and demonstrated that
length variations among genotypes were caused by insertion–
deletion (indel) polymorphisms within an intron. Similarly,
Hongtrakul et al. (1998) identified intron-length polymorphism
in sunflower (Helianthus annus L.) caused by indels
and differences in lengths of monomeric repeats, and suggested
their utility as allele-specific DNA markers. Intronlength
variation due to transposable-element indels in
introns has also been discovered among alleles of maize
genes (Bureau and Wessler 1994; Esen and Bandaranayake
1998).
The relative levels of allelic variability in lengths of
introns, exons, or other gene regions have not been adequately
studied in any species (Long and de Souza 1998).
We expect that the phenotypic consequences of length variation
are not equal among gene regions. Sequences that have
large and direct effects on phenotypes are likely to maintain
the least amount of variation under selection. Therefore, we
expect exon sequences to have lower levels of length polymorphism,
but the relative levels of variation among exons,
introns, and upstream regions are not predictable because
introns as well as promoter regions can affect gene expression
(Bolle et al. 1996; Long and de Souza 1998). This implies
that not all variation within introns and promoter
regions is neutral. Introns contain the splice sites that direct
their correct removal from the gene when initial transcripts
are processed to mature RNAs (Lewin 1997). If these sites
are changed or removed, the gene product may be altered,
resulting in a nonfunctional protein (Brown et al. 1996).
Laurie and Stam (1994) determined that polymorphism
within an intron of the alcohol dehydrogenase gene in
Drosophila melanogaster has an effect on the amount of
protein present. Similarly, Fridman et al. (2000) suggested
that length variation within the intron of an invertase gene in
tomato was responsible for allelic differences in the gene’s
expression. Furthermore, intron size itself seems to be constrained
within maximal and minimal limits by selection
pressures (Carvalho and Clark 1999). The importance of
promoters in gene expression is obvious, but it is not known
to what extent variation within a few hundred base pairs (bp)
upstream of the transcription start site will affect expression.
Empirical evidence regarding the relative levels of variation
among these different gene regions is needed to better understand
the phenotypic importance of different gene regions
and predict the feasibility of developing DNA markers by
amplifying particular gene regions.
The objectives of this study were to develop PCR primer
pairs targeting previously sequenced genes from Avena and
maize in order to compare the amount of allelic amplification
product polymorphism among gene regions. Exon, intron,
promoter, and SSR target regions were compared in maize,
while SSR and intron target regions were compared in oat.

15. Vigouroux, Y., Jaqueth, J. S., Matsuoka, Y., Smith,

O. S., Beavis, W. D., Smith, J. S. C. &Doebley, J. (2002)
Mol. Biol. Evol., in press“Identifying genes of agronomic
importance in maize
by screening microsatellites for evidence of
selection during domestication”
16. 3 Remington, D. L., Thornsberry, J. M., Matsuoka, Y.,
Wilson, L. M., Whitt, S. R., Doebley
17.J., Kresovich, S., Goodman, M. M. & Buckler, E. S. (2001)
Proc. Natl. Acad. Sci. USA 98,
11479–
18. ‘’Genetic Structure and Diversity among Maize Inbred Lines as Inferred from
DNA
Microsatellites “”Department of Statistics, North Carolina State University,
Raleigh, NC 27695,
2Department of Crop Science, North Carolina State University, Raleigh, NC 27695,
3Crop Genetics Research and Development, DuPont Agriculture and Nutrition, Pioneer
Hi-Bred
International, Johnston, IA 50131,
4USDA-ARS and Department of Genetics, North Carolina State University, Raleigh, NC
27695,
5Laboratory of Genetics, University of Wisconsin, Madison, WI 53706

19. “SSR marker based DNA fingerprinting and diversity

study in rice (Oryza sativa. L)”
B. Kalyan Chakravarthi1 and Rambabu Naravaneni2
20. “The efficiency of SSR markers in genetic
diversity estimation and gene pool classification of
common bean (Phaseolus vulgaris L.)” Marko MARAS1, Jelka ŠUŠTAR-
VOZLIČ2, Branka JAVORNIK3, Vladimir M

L1 L2 L3 L4 L5 L6 L7

M1 152 183 223 245 188 200,234 122,202

M2 152 200 223,264 245 202 200,234 122,202
M3 144 172 223,249 200,262 171 190 122,202
M4 152 183 249,273 200,262 188 190,224 122,202
M5 153 178,212 235 195 188 190 122,202
EGLIČ4