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(name of student)
(student number)
(Program)
Semester 5, Work Term 1
(date)
Page 2
(Address)
(Date)
My first work term in Summer ’01 was spent in “name of company”. I worked in
the Molecular Biology Department under the supervision of “name of supervisor”.
Aside from these goals, I was also able to gain experience in many other techniques of
molecular biology and Marker assisted selection (MAS) in plant agriculture. This report
will provide a general overview of Marker Assisted Selection in plant breeding.
This report has more of a review format then of primary literature. The reason for
this is that plant breeding programs are often very long in duration and it can take several
months to fully complete one. I was not present from start-to finish of any single
program. But, I have included a short summary of one the experiments that was being
performed during my work term.
Sincerely
(name of student)
Page 3
(name of student)
(student number)
(Program)
Semester 5, Work Term 1
“name of company”
“Date”
Page 4
Table of Contents
Title Page
Cover Page 1
Letter of Submital 2
Title Page 3
Table of Contents 4
Introduction 5
Types of Markers 6
i) Morphological Markers 6
ii) Biochemical Markers 6
iii) Molecular Markers 7
Genetic Mapping and Linkage Analysis 8
Gel Electrophoresis 11
Polymerase Chain Reaction 13
Molecular Marker Techniques 15
i)Restriction Fragment Length Polymorphism (RFLP) 15
ii)Randomly Amplified Polymorphic DNA Markers (RAPD) 18
iii)Simple Sequence Repeat (SSR)/Microsatellites 18
iv) Amplified Fragment Length Polymorphism 20
Development and Characterization of Simple Sequence Repeat (SSR) 22
Markers and Their Use in Detecting Relationships Among Tomato
Cultivars.
Introduction 22
Materials and Methods 23
Results 25
Discussion 27
Conclusion 27
Acknowledgments 28
References 29
Bibliography 30
List of Illustrations
Figure 1: Sequential Summary of events occurring in isozymes analysis 7
Figure 2: Crossing over of chromosomes in Anaphase of meiosis 8
Figure 3: Construction of mapping population 9
Figure 4: An example of chromosome map 10
Figure 5: Polymerase Chain Reaction 13
Figure 6: Summary of Major events in RFLP analysis 17
Figure 7: Summary of major events in SSR analysis 20
Figure 8: Summary of major events in AFLP analysis 22
Figure 9: Banding Patter of AI778183 primer in 6% polyacrylamide gel 25
with silver staining
Figure 10: Banding pattern obtained from primer AI 895937 26
Figure 11: Banding pattern obtained from AW037347 26
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Introduction
Within the last twenty years, molecular biology has revolutionized conventional breeding
techniques in all areas. Biochemical and Molecular techniques have shortened the
duration of breeding programs from years to months, weeks, or eliminated the need for
them all together. The use of molecular markers in conventional breeding techniques has
also improved the accuracy of crosses and allowed breeders to produce strains with
combined traits that were impossible before the advent of DNA technology (1).
appearance, yield, and disease resistance. This is performed with the assistance of
identifiable traits. When a detectable mutant is identified within a population, the gene
causing the mutation was placed on a genetic map through a series of crosses that would
establish its recombination frequency relative to other genes that had previously
discovered and mapped. If the mutant gene was in close proximity to the gene for a
desired trait, the mutant gene or “marker” was said to be linked to it because the marker
and the gene tend to co-segregate. In a breeding cross, this mutant gene could be used to
detect whether or not a breeding cross had been successful in transferring the desired
trait. If the mutant gene is observed being expressed in the progeny, it is most likely that
the progeny also has the desired trait due to its link to the mutant gene. This is the
phenomenon of co-inheritance and the selection of these mutant genes for the tracking of
This breeding technique can be used in almost any species: animal, plant, fungi or
bacteria but the focus here will be on marker assisted selection in agriculture. This paper
will cover how DNA technology has improved upon these techniques and eliminated
Types of Markers
i) Morphological Markers
These are the traditional markers mentioned before. Morphological mutant traits
and indirect selection is carried out using the physically identifiable mutant for the trait.
There are several undesirable factors that are associated with morphological markers.
The first is there high dependency on environmental factors. Often the conditions that a
plant is grown in can influence the expression of these markers and lead to false
determination. Second, these mutant traits often have undesirable features such as
dwarfism or albinism. And lastly, performing breeding experiments with these markers
is time consuming, labour intensive and the large populations of plants required need
Isozymes are used as biochemical markers in plant breeding. Isozymes are common
enymes expressed in the cells of plants. The enymes are extracted, and run on denaturing
electrophoresis gels. The denaturing component in the gels (usually SDS) unravels the
secondary and tertiary structure of the enzymes and they are then separated on the basis
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of net charge and mass. Polymorphic differences occur on the amino acid level allowing
marker.
Fig.1: Sequential
Summary of events
occurring in isozymes
analysis of plant
samples. Although
varieties, isozymes
Biochemical markers are superior to morphological markers in that they are generally
MAS is that most cultivars (commercial breeds of plants) are genetically very similar and
protein primary structure may still cause an alteration in protein function or expression.
sequences (i.e.: base pair deletions, substitutions, additions or patterns) (4). There are
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various methods to detect and amplify these polymorphisms so that they can be used for
breeding analysis and these techniques will be the focus of this paper. Molecular markers
are superior to other forms of MAS because they are relatively simple to detect, abundant
environmental conditions and can be detected at virtually any stage of plant development.
1) Must be polymorphic
2) Co-dominant inheritance
5) Reproducible
Molecular markers can be used for several different applications including: germplasm
but with far more advanced techniques. The basis of genetic mapping is the phenomenon
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exchange sections of their gene sequence. The tendency of two genes to recombine is
used as a measurement of their linkage and distance on a genetic map. For example, two
not, cross-over and form recombinants. The expected ratio of phenotypes for the F2
population is the classic Mendelian 1:2:1, it is the divergence from this ratio that
determines the amount of linkage between genes. For example of there are two markers
X and Y which are co-dominant for parents A and B; parent A is homozygous (XX YY)
and parent B is homozygous (xx yy). Meiosis in the parents will produce gametes (XY)
in A and (xy) in B, therefore the F1 cross will produce a hybrid (Xx Yy). Gamete
formation in the hybrid is when crossing over becomes important. There are four
possibilities: The first two are the original or “parental” genotypes (XY) and (xy), the
second two are (Xy) and (xY) which are the “recombinant” genotypes. These are called
the recombinant genotypes because their formation is only possible through crossing over
if no recombination was occurring. With classic, independent assortment, the ratio would
be:
13 (XY XY) : 12 (Xy Xy) : 25(XY xY) : 25 (XY Xy) : 50 ( XY xy) : 25 (xY xy) : 25 (Xy xy) : 13 (xY xY) : 12 (xy xy)
Notice all of the recombinant gametes are in bold. To determine the recombination
Note that all progeny with two recombinant gametes are counted twice because they
contain two recombinant gametes. There for the recombination frequency for this
This is the upper limit of recombination frequency in a selfed F2 population meaning that
alleles X and Y are at opposite ends of the same chromosome, any recombination
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frequency lower than this indicates that they genes are linked to some degree. Thus,
genes are mapped on to a chromosome relative to each other through mounds and
specific trait.
Gel Electrophoresis
The technique finds its roots in chromatography and its basis is that molecules with a net
charge will move through an electric field and their progress will be retarded to varying
degrees depending on the matrix in which they are moving. The velocity of migration
depends on the strength of the electric field, net charge on the molecule and the frictional
f = frictional co-efficient
Gel is almost exclusively used as the electrophoresis medium because of its ability to
suppress small temperature gradients which can cause fluctuations in current. It’s also
easy to work with and handle and its concentration can be varied to optimize separation.
Two types of gels are commonly used. Polyacrylamide was used first and can
attain high resolution of extremely small molecular weight differences. It does present
some problems however; in its unpolymerized form it is a potent neurotoxin and requires
extract from seaweed that boils at approx 94 degrees Celsius and when cools, forms a
tight gel matrix. It cannot resolve as well as polyacrylamide, but it is inexpensive and
safe enough to eat. Metaphor agarose is a highly pure form of agarose that, at high
DNA from comparatively small amounts. It was developed during 1985-1986 by the
Cetus Corporation as an in vitro method of DNA amplification. The process involves the
oligonucleotide primers to
Initially conventional E.coli DNA polymerase was used, but it is not stable at 95 degrees
and new polymerase had to be added after each denaturation cycle. This was all changed
with the discovery of Taq (Thermus aquaticus – a bacteria found at the opening of
thermal vents of the ocean floor) polymerase by Kary Mullis which was stable at the
denaturation temperature and could be used throughout the entire process without having
4) Target DNA sequence. Purity is not a huge problem as long as pH/Taq okay
These components are mixed together with double distilled/autoclaved water and put into
This cycle is repeater 30-40 times before completion. Taq polymerase has no 5’-3’
exonuclease proofreading activity and thus can be prone to errors and these errors are
propagated with each new cycle; this why some manufactures often sell Taq mixed with a
This was the first molecular marked technique developed and used in MAS for
plant breeding. The technique centers around the digestion of genomic DNA digested
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with restriction enzymes. These enzymes are isolated from bacteria and consistently cut
DNA at specific base pair sequences which are called recognition sites. These
recognition sites are not associated with any type of gene and are distributed randomly
throughout the genome. When genomic DNA is digested with one of these restriction
enzymes, (of which there are thousands, each cutting at a specific sequence), a series of
fragment are produced of varying length. These fragments are separated using agarose or
DNA has a uniform charge per unit length when run under electrophoresis
conditions which arises from the phosphates groups in its backbone. So when DNA
fragments are separated via electrophoresis, the distance they travel is dependent only on
their molecular weight. This allows their molecular weight to be determined with simple
standard called DNA ladders which are run along side the DNA in the gel. When
restriction fragments are separated on agarose gels a series of bands results. Each band
corresponds to a restriction fragment of different length. The lighter they are the farther
pair deletions, mutations, inversions, translocations and transpositions which result in the
polymorphism.
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Only a single base pair difference in the recognition site will cause the restriction enzyme
not to cut. If the base pair mutation is present in one chromosome but not the other, both
fragment bands will be present on the gel, and the sample is said to heterozygous for the
marker. Only co-dominant markers exhibit this behavior which is highly desirable,
dominant markers exhibit a present/absent behavior which can limit data available for
analysis (2).
1) DNA isolation – a significant amount of DNA must be isolated from the sample
and purified to a fairly stringent degree as contaminants can often interfere with
the restriction enzyme and inhibit its ability to digest the DNA.
buffered conditions. The enzyme cuts at recognition sites throughout the genome
3) Gel electrophoresis – The digest is run on a gel and when visualized appears a
constructed to visualize more specific bands in the digest. These probes consist of
6) Analysis
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To develop probes to screen RFLP, an initial digest must be performed on the species of
interest. The restriction fragments are ligated into a plasmid vector and transformed into
bacteria. Positive cultures are then isolated, ligated sequences removed, amplified by
RAPD was the first PCR based molecular marker technique developed and it is by
far the simplest. Short PCR primers (approximately 10 bases) are randomly and
arbitrarily selected to amplify random DNA segments throughout the genome. The
priming sites in the appropriate orientation. RAPD often shows a dominant relationship
due to primer being unable to bind (show 3:1 ration, unable to distinguish between
homozyogotes and heterozygotes) (5). RAPD products are usually visualized on agarose
Simple sequence repeats are present in the genomes of all eukaryotes and consists
Mono: A, T
Di: AT, GA
Tri: AGG
Tetra: AAAC
These repeated motifs are denoted (AAAC)n, where n is the number of tandem repeats.
The sequences flanking these microsatellites are often conserved and can be used to
design primers. These primers can be designed by constructing a novel genomic library
and sequencing segments of the subject genome. Already discovered sequence (i.e.:
GENEBANK online database) can also be searched for SSRs and primers designed from
that. Polymorphism is based on the number of tandem repeats and therefore the length of
the PCR products. SSR is a co dominant marker such as RFLP and is usually visualized
AFLP is the latest form of marker assisted selection and is a highly sensitive
method based on the combined concepts of RFLP and RAPD. This technique is
applicable to all species giving very reproducible results. The basis of AFLP is the PCR
1) DNA is cut with two specific restriction enzymes, one frequent cutter (3 bp
2) Oligonucleotide “adapters” are ligated to the ends of each fragment. One end
with a complimentary sequence for the rare cutter and the other with the
complimentary sequence for the frequent cutter. This way only fragments
which have been cut by the frequent cutter and rare cutter will be amplified.
3) Primers are designed from the known sequence of the adapter, plus 1-3
selective nucleotides which extend into the fragment sequence. Sequences not
4) PCR performed
Typical results give 50-100 bands despite selective nucleotides and rare/frequent
selection. This high number of bands eases analysis by providing more chance of
polymorphism (4).
Introduction
experiment SSRs are used to compare 19 cultivars of tomato from various geographic
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locations around the world and asses their genetic proximity to one another. Another
outcome to this experiment is the discover of several novel SSR markers to contribute to
SSRs were chosen due to their abundance in the tomato genome and the wealth of
Primer Design
Primers were designed from conserved flanking sequences obtained from online
databases such as GENEBANK. They were on average 18-24 base pairs in length with a
genomic DNA, 5 Units of Taq polymerase, 0.2mM each dNTPs, 10x PCR reaction buffer
containing MgCL2, and H2O to volume. Reactions were performed on a heated lid
thermal cycle for an initial denaturation step of 94 degrees C for 5 minutes; followed by
Cultivars
1 Borbas Hungaria
4 Cocabul France
13 S-11-83-4 China
14 Saljut Russia
16 Scorpio Australia
17 White Fruit ?
18 DRS-Ben Holland
19 DRS-Bosch Holland
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Analysis
Gels were visualized on 6% polyacrylamide and silver stained. The gels were run for
Results
500 tomato DNA sequences were searched for SSRs and analyzed, 158 primer pairs were
designed and purchased. 127 of these were screened of which approx 45% showed
polymorphism.
Below (Figure 9) is shown the banding pattern of the AI778183 primer. There
are three distinct band morphologies, (A) which is the upper most orientation
characterized by the 2nd band from the left, (B) which is characterized by the leftmost
band, and the heterozygote of which there is one example 7 bands from the left.
Fig 9: Banding Patter of AI778183 primer in 6% polyacrylamide gel with silver staining.
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Fig 10 was obtained from primer AI895126 and only has two distinct
morphologies (A) which is the upper most band and (B) which is the lower band. The
absence of heterozyogtes in this figure does not necessarily indicate that the primer is
unable to express them. Often in tomato cultivars, heterozygosity has been bread out of
their genome.
Fig 1.3 was obtained from primer AW037347 and also has two distinct
morphologies. (A) which is the topmost band, (B) which is the band. This primer is
Discussion
Although I was not present to witness the end of the experiment, sufficient data
was accumulated to indicate that a phylogentic analysis would be possible. Fig 12.4
As seen on the tree, the two cultivars obtained form Holland have the closest
genetic relationship, which is to be expected since they originated from the same
breeding program. Common ancestors then branch out in no predictable fashion. This is
into their breeding plan. This will provide diversity and increase the likelihood of a
Conclusion
From the data obtained, SSRs seem to be a very useful tool in analyzing the
plant breeding. Through these techniques, plant breeders have been able to produce
cultivars of agriculturally significant plants with genes for resistance to many diseases
untrue. MAS is simply an improvement on an age old method of improve plant quality
and yield. No foreign DNA is introduced into the plant, and no environmentally harmful
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genes have been incorporated. MAS is simply the transfer of useful traits among already
In future research, the genetic maps that have been developed by MAS will
become more and more saturated as more techniques are developed and more markers
uncovered and mapped. This technique, once normalized will provide small scale plant
breeders to compete with such giants as Monsanto and Pioneer in the race to produce
Acknowledgements
I would like to thank XXX and all of the staff in the molecular biology
department of the XXX, Ontario, for all of there tutelage and help over my first work
term.
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References
1) Stuber, C. W., Polacco, M., Senior, M.L. (1999) Synergy of Empirical Breeding,
Marker Assisted selection and Genomics to Increase Crop Yield Potential, Crop
Science 39:1571-1583
2) Yu, Y.G., Saghai-Maroof, M.A., Buss, G.R., Maghan, P.J., Tolin, S.A., (1993)
3) Huang, C.C., Cui, Y.Y., Weng, C.R., Zabel, P., (2000) Development of diagnostic
PCR markers closely linked to the tomato powdery mildew resistance gene OI-1
918-924
4) Gupta, P.K., Varshney, R.K., Sharma, P.C., Ramesh, B., (1999) Molecular
Markers and their applications in wheat breeding; Plant Breeding 118: 369-390
5) Yin, T., Huang, M., Wang, M., Zhu, L., Zeng, Z., Wu, R (2001) Preliminary
6) Senior, M.L., Chin, E.C.L., Lec, M., Smith, J.S.C., Stuber, C.W (1996) Simple
Bibliography
Enfield, USA
2) Winfrey, Rott, Wortman, (1997) Unravelling DNA: Molecular Biology for the
3) Sunstad, Simmons, Principles of Genetics, 2nd Edition, John Wiley & Sons, Inc.,