Marker Assisted Selection Techniques In Plant

Page 1

Review of Marker Assisted Selection techniques in Plant Breeding

(name of student) (student number) (Program) Semester 5, Work Term 1 (date)

Page 2 (Address) (Date) Dr. William Tam Faculty Advisor Department of Chemistry and Biochemistry University of Guelph Guelph, ON N1G 2W1 Dear Dr. Tam 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”. The achievements of my work term were as follows: 1) Run SSR analysis on tomatoes using PCR and Polyacrylamide Gel Electrophoresis (PAGE) to discover novel molecular markers for genome mapping. 2) Develop mapping population by performing interspecific crosses among cultivars and wild tomato breeds. 3) Maintain current breeding population and sample DNA for analysis as needed. 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. Lastly, primer sequences have been excluded due to proprietary needs. Sincerely (name of student)

Page 3

Review of Marker Assisted Selection techniques in Plant Breeding

(name of student) (student number) (Program) Semester 5, Work Term 1

“name of company” “Date”

Page 4 Table of Contents Title Cover Page Letter of Submital Title Page Table of Contents Introduction Types of Markers i) Morphological Markers ii) Biochemical Markers iii) Molecular Markers Genetic Mapping and Linkage Analysis Gel Electrophoresis Polymerase Chain Reaction Molecular Marker Techniques i)Restriction Fragment Length Polymorphism (RFLP) ii)Randomly Amplified Polymorphic DNA Markers (RAPD) iii)Simple Sequence Repeat (SSR)/Microsatellites iv) Amplified Fragment Length Polymorphism Development and Characterization of Simple Sequence Repeat (SSR) Markers and Their Use in Detecting Relationships Among Tomato Cultivars.

Page 1 2 3 4 5 6 6 6 7 8 11 13 15 15 18 18 20 22

Introduction Materials and Methods Results Discussion Conclusion Acknowledgments References Bibliography

22 23 25 27 27 28 29 30

List of Illustrations Figure 1: Sequential Summary of events occurring in isozymes analysis Figure 2: Crossing over of chromosomes in Anaphase of meiosis Figure 3: Construction of mapping population Figure 4: An example of chromosome map Figure 5: Polymerase Chain Reaction Figure 6: Summary of Major events in RFLP analysis Figure 7: Summary of major events in SSR analysis Figure 8: Summary of major events in AFLP analysis Figure 9: Banding Patter of AI778183 primer in 6% polyacrylamide gel with silver staining Figure 10: Banding pattern obtained from primer AI 895937 Figure 11: Banding pattern obtained from AW037347

7 8 9 10 13 17 20 22 25 26 26

Page 5 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).

Breeding is simply defined as the selective mating of individuals of a population to isolate or combine desired morphological, physiological or genetic traits such as 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 desired traits is called indirect selection.

Page 6 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 most of the error involved with them.

Types of Markers i) Morphological Markers These are the traditional markers mentioned before. Morphological mutant traits in a population are mapped and linkage to a desirable or undesirable trait is determined 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 large plots of land and/or greenhouse space in which to be grown (1).

ii) Biochemical Markers 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

Page 7 of net charge and mass. Polymorphic differences occur on the amino acid level allowing singular peptide polymorphism to be Detected and utilized as a polymorphic biochemical marker. Fig.1: Sequential Summary of events occurring in isozymes analysis of plant samples. Although useful in some plant varieties, isozymes provide little variation in highly bred cultivars.

Biochemical markers are superior to morphological markers in that they are generally independent of environmental growth conditions. The only problem with isozymes in MAS is that most cultivars (commercial breeds of plants) are genetically very similar and isozymes do not produce a great amount of polymorphism and polymorphism in the protein primary structure may still cause an alteration in protein function or expression.

iii) Molecular Markers Molecular markers are based on naturally occurring polymorphisms in DNA sequences (i.e.: base pair deletions, substitutions, additions or patterns) (4). There are

Page 8 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 throughout the genome even in highly bred cultivars, completely independent of environmental conditions and can be detected at virtually any stage of plant development. There are 5 conditions that characterize a suitable molecular marker (4): 1) Must be polymorphic 2) Co-dominant inheritance 3) Randomly and frequently distributed throughout the genome 4) Easy and cheap to detect 5) Reproducible Molecular markers can be used for several different applications including: germplasm characterization, genetic diagnostics, characterization of transformants, study of genome organization and phylogenic analysis.

Genetic Mapping and Linkage Analysis The

techniques

of

genetic

mapping and linkage analysis were developed in 1911 by D.H Morgan and his graduate student Alfred H. Sturtevant and are still used today in much the same way but with far more advanced techniques. The basis of genetic mapping is the phenomenon

Page 9 of “crossing-over” of chromosomes during meiosis, where homologous chromosomes 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 genes that recombine often are far apart on a genetic map and two that rarely recombine are said to be “linked” and are very close together on a genetic map. To determine trait recombination frequencies and form a genetic map, a mapping population must first be produced.

The first step in producing a

mapping population is selecting two genetically divergent parents (that will still produce viable progeny). Often one common cultivar and one wild parent are selected as they are likely to be the most divergent. The two selected parents are screened for polymorphism with the markers that are to be mapped to be sure that the progeny will produce recombinants. The mapping population is then produced by crossing the two parents to form an F1 hybrid population which is selfed to produce an F2 population which can be used for mapping(4).

The initial cross will produce a

uniform, heterozygous population with each plant contain one chromosome from each parent.

During

meiosis, the homologous chromosomes may or may

Page 10 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 of the homologous chromosomes. In a population of 200 of these F2 plants an expected ratio would be: 50 (XY XY) : 100 (XY xy): 50 (xy xy)

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 frequency, the number of recombinant gametes is divided by total number of gametes. Note that all progeny with two recombinant gametes are counted twice because they contain two recombinant gametes.

There for the recombination frequency for this

population is 150/400 = 0.375 and are said to be 37.5 centiMorgans apart. 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

Page 11 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 mounds of data. This process is now performed almost exclusively by computer programs (3). Once a genetic linkage map of a chromosome has been established, gene locations which have been mapped in a similar manner can be integrated into the map. The genes can then be assessed for linkage with the closest markers on the map and indirect selection performed using them. For a chromosome map to be useful, it must be saturated with markers of several different types so that a variety of markers can be tried and assessed for their usefulness in detecting that specific trait.

Gel Electrophoresis A common technique used in the analysis of molecular markers is 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 co-efficient of the particle in the matrix.

Page 12 E = magnitude of the electric field v=

Ez f

z = net charge of the particle f = the frictional co-efficient

f = frictional co-efficient f = 6πηr

n = medium viscosity r = radius of the particle

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 a great amount of care in handling and must be disposed of appropriately. Agarose is an 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 concentrations can achieve highly resolved separations of heavy molecules separated under high electric field conditions.

Page 13 Polymerase Chain Reaction (PCR) PCR is a method of selectively or non-selectively producing large amounts of 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 denaturation of the target DNA at 95 degrees followed by the annealing of oligonucleotide

primers

to

sequences flanking the target DNA for amplification which allow DNA polymerase to bind and begin synthesizing novel DNA. This cycle is repeated over and over again, each time doubling the amount of DNA present.

After 30 cycles, the

final amount of DNA will be 230 times the original amount. 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 to add any more. To perform PCR the following components are needed:

Page 14 1) Forward and reverse oligonucleotide primers 2) Amplification buffer (KCl, TrisCl, 1.5mM MgCl2) this is to control the pH drop when incubated at the extension step. 3) dNTPs at saturation concentration (can be less) 4) Target DNA sequence. Purity is not a huge problem as long as pH/Taq okay 5) Taq DNA polymerase. These components are mixed together with double distilled/autoclaved water and put into a thermal cycler which adjusts the temperature in the following sequence. 1) Denaturation of template DNA at 94◦C 2) Annealing of primers to target sequences at 35-65◦C 3) DNA synthesis from 3’ end of each primer by Taq Polymerase 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 thermally stable exonuclease to perform this function.

Molecular Marker Techniques i) Restriction Fragment Length Polymorphism (RFLP) 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

Page 15 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 polyacrylamide gel electrophoresis (PAGE) and yield a characteristic pattern. 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 they have traveled. Variations in the characteristic pattern of a RFLP digest can be caused by base pair deletions, mutations, inversions, translocations and transpositions which result in the loss or gain of a recognition site resulting in a fragment of different length and polymorphism.

Page 16

Page 17 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).

Procedure for RFLP 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. 2) Restriction Digest - Restriction enzyme is added to purified genomic DNA under buffered conditions. The enzyme cuts at recognition sites throughout the genome and leaves behind hundreds of thousands of fragments. 3) Gel electrophoresis – The digest is run on a gel and when visualized appears a smear because of the large number of fragments. 4) Transfer to nitrocellulose membrane filter 5) Probe visualization – Because of the large number of fragments, probes must be constructed to visualize more specific bands in the digest. These probes consist of radio labeled oligonucleotide sequences which will anneal to the fragment sequences so that that they may be visualized on photographic paper using a technique called a southern blot. 6) Analysis

Page 18

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 PCR and radio-labeled for use as RFLP probes.

PCR Based Molecular Markers i) Randomly amplified polymorphic DNA Markers (RAPD) 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 resulting amplification product is generated at the region flanking a part of the 10 bp 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 gels stained with ethidium bromide.

ii) Simple Sequence Repeats (SSR)/Microsatellites Simple sequence repeats are present in the genomes of all eukaryotes and consists of several to over a hundred repeats of a 1-4 nucleotide motif.

Page 19

Page 20 Some common motifs are: 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 on metaphor agarose or polyacrylamide gels (6).

iii) Amplified Fragment Length Polymorphism (AFLP) 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 amplification of restriction enzyme fragments of genomic DNA.

Page 21

Page 22 1) DNA is cut with two specific restriction enzymes, one frequent cutter (3 bp recognition site) and one rare cutter (6 bp recognition site). 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 matching these selective nucleotides in the primer will not be amplified. 4) PCR performed 5) Visualized on agarose gels with ethidium bromide 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).

Development and Characterization of Simple Sequence Repeat (SSR) Markers and Their Use in Detecting Relationships Among Tomato Cultivars.

Introduction As explained previously molecular makers can be used to perform phylogenic analysis on a species by comparing the presence/absence of various markers in their genome. In this experiment SSRs are used to compare 19 cultivars of tomato from various geographic

Page 23 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 the overall genetic map of the tomato SSRs were chosen due to their abundance in the tomato genome and the wealth of tomato genomic DNA sequence available online to aid in primer design.

Materials and Methods 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 melting temp of 47 degrees C.

PCR amplification Conditions Samples reaction volumes were 10 microlitres consisting of 0.3pM of primer, 2.5nM of 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 30 cycles of: 25 secs @ 92 degrees C (denaturation), 25 secs @47-60 (annealing) degrees C and 25 secs@ 68 degrees C (extension).

Page 24 Cultivars No.

Name

Origin

1

Borbas

Hungaria

2

Bulgaria 436-76

Bulgaria

3

Cc218

ON, Canada

4

Cocabul

France

5

Cornell-1010

NY, USA

6

FM 6203

CA, USA

7

Heinz 916010

On, Canada

8

L2024

South Africa

9

N1190

ON, Canada

10

NC EBR-111

NC, USA

11

Ohio 8245

OF, USA

12

Purdue 812

IN, USA

13

S-11-83-4

China

14

Saljut

Russia

15

Sandpoint

Or, USA

16

Scorpio

Australia

17

White Fruit

?

18

DRS-Ben

Holland

19

DRS-Bosch

Holland

Page 25 Analysis Gels were visualized on 6% polyacrylamide and silver stained. The gels were run for approximately 1 hour and 45 minutes at 60W and stained immediately afterward.

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.

Page 26 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 another

example

of

a

dimorphic

Fig 10: Banding battern obtained from primer AI 895937

Fig 11: Banding pattern obtained from AW037347

primer.

Page 27

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 shows a preliminary phylogentic tree based on the data currently accumulated. 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 a result of tomato breeders obtaining resources from a variety of locations to incorporate into their breeding plan. This will provide diversity and increase the likelihood of a breeding ending up with a resistant tomato line.

Conclusion From the data obtained, SSRs seem to be a very useful tool in analyzing the genetic relationship among cultivated species such as tomatoes.

They provide

reproducible results and are fairly simple to obtain. Overall, marker assisted selection has proven to be a very useful technique in plant breeding. Through these techniques, plant breeders have been able to produce cultivars of agriculturally significant plants with genes for resistance to many diseases that were not possible before the advent of DNA technology. One common miss conception is that MAS is a form of transgenics. This is 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

Page 28 genes have been incorporated. MAS is simply the transfer of useful traits among already potential mating population. 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 cultivars with broad based resistance to disease.

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.

Page 29 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) RFLP and Microsatellite Mapping of a Gene of Soybean Mosaic Virus Resistance, Phytopathology 84: 60-64 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 on chromosome 6 of tomato; Journal of Theoretical and Applied Genetics 101: 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 interspecific genetic maps of the Populus genome constructed from RAPD markers; Genome 44: 602-609 6) Senior, M.L., Chin, E.C.L., Lec, M., Smith, J.S.C., Stuber, C.W (1996) Simple Sequence Repeat Markers Developed from Maize Sequences Found in the GENBANK Database: Map Construction; Crop Science 36: 1676-1683

Page 30 Bibliography

1) Chawla, H.S., (2000) Introduction to Plant Biotechnology; Science Publishers Inc, Enfield, USA 2) Winfrey, Rott, Wortman, (1997) Unravelling DNA: Molecular Biology for the Laboratory; Prentice Hall, Upper Saddle River, NJ, USA 3) Sunstad, Simmons, Principles of Genetics, 2nd Edition, John Wiley & Sons, Inc., New York, USA 4) Wetermeier, Reiner (1993) Electrophoresis in Practice; VCH, New York, USA 5) Hawcroft, D.M., The Basics of Electrophoresis, IRL Press, Oxford, England