Gene mapping, describes the methods used to identify the locus of a gene and the distances between genes. The essence of all genome mapping is to place a collection of molecular markers onto their respective positions on the genome. Molecular markers come in all forms.

1. GENE MAPPING & MOLECULAR
MAPS OF PLANT GENOME
ANURAG RAGHUVANSHI
Dept. of Pharmacology
anuragraghuvanshi3@gm
ail.com

2. CONTENTS
* PLANT CHROMOSOME ANALYSIS
* MAPPING OF PROKARYOTIC &
EUKARYOTIC GENES.
* USES OF PCR IN GENE MAPPING
* MOLECULAR MPS-RFLP, RAPD AND
THEIR APPLICATION ON
DETECTION OF ADULTERANTS
* PHYSICAL MAPS IN-SITU
HYBRIDIZATION

3. INTRODUCTION TO PLANT CHROMOSOME
ANALYSIS
• Chromosome analysis and sorting using flow cytometry (flow cytogenetics) is an
attractive tool for fractionating plant genomes to small parts. The reduction of
complexity greatly simplifies genetics and genomics in plant species with large
genomes.
• During the past decade, significant progress has been made in the development of
methods for the preparation of plant chromosome suspensions suitable for flow
cytometric analysis.
• sorted chromosome were used for the establishment of chromosome specific DNA
libraries & for gene mapping.

4. TECHNIQUES FOR QUANTITATIVVE
CHROMOSOME ANALYSIS
CYTOPHOTOMETRY
CYTOFLUROMETRY
IMAGE ANALYSIS

5. TYPES OF FLOW CHAMBER IN FLOW
CYTOMETERS

6. PRINCIPLE USED IN FLOW SORTERS

7. PREPARATION OF CHROMOSOME SUSPESION
• The problem associated with preparation of high quality suspension of plant
chromosomes may be considered the main reason for the delay in flow cytometric
analysis and sorting of plant chromosomes,
• An ideal chromosome suspension should contain intact & well-dispersed chromosomes
and should be free of contaminating cellular debris.
• This is difficult to achieve with plant material for several reasons:
1. It is not easy to obtain high degree of mitotic synchrony in plant tissues
2. Chromosome stickiness and clumping is often observed after treatment with metaphase
blocking agents.
3. Plant chromosome blocked in metaphase have tendency to split into single chromatids.
4. The present of a rigid cell wall makes the release of chromosomes more difficult in
comparisons with human or animal calls
5. Plant chromosomes are not stable for longer periods in currently used isolation buffers.

8. ISOLATION OF CHROMOSOME
1. INDUCTION OF CELL CYCLE SYNCHRONY
2. ACCUULATION OF CELLS IN METAPHASE
3. RELEASE OF CHROMOSOMES

9. CHROMOSME ANALYSIS
• UNIVARIATE FLOW KARYOTYPING
• BIVARIATE FLOW KARYOTYPING
• With most od the commercially available flow cytometers, wo basic optical parameters of particles can be
analysed:
1. Light scatter
2. Fluorescence emission
• Chromosome morphology( length, shape) may vary considerably among the population of chromosomes of
the same type, which is reflected by broad distribution of light scatter intensity. This does not permit
discrimination of single chromosome types.
• On the other hand DNA content is essentially constant for each chromosome type, and thus fluorescence
intensity related to the chromosomal DNA contents has been used as a basic parameters in flow
cytometric analysis of isolated chromosome since its early beginnings (Carrano et. Aa.)

10. CHROMOSOME STAINING
1. INTERCALATING DYES
2. DNA-SPECIFIC DYES
3. FLUORESCENT ANTIBIOTICS
4. DYE COMBINATIONS

11. ADVANTAGE & FUTURE OF FLOW CYTOMETRY
• As yet, flow cytometry is not a major technique in plant chromosome analysis.
however, its advantage in term of speed, accuracy, and quality of quantitative
measurement make the technique one which likely to become more widespread.
• While nuclear DNA measurement can be quickly obtained, & are of value in
examining purity of plant stocks, keening for aneuploidy or polyploidy, flow
karyotyping is considerably behind animal work.
• Because of quantitative measurements which can be made, flow cytometry can be
used for checks and quality control of many aspects of plant genetics,. In plant
breeding and seed production, the ability to qualify nuclear size and hence assess
purity and ploidy is likely to be useful.

12. BIVARIATE FLOW KARYOTYPES OF VICIA FABA,
S. LUCRETTI AND J. DOLEZEL
• A) Fluorescence pulse area (FPA) and fluorescence pulse
width (FPW) are used simultaneously as parameters.
Groups corresponding to six pairs of chromosomes
(higher values of FPW, similar values of FPA) are
resolved. Chromatid doublets of different chromosomes
are shown also.
• B) Contour plot of MI/DAPI stained field bean
chromosomes. The chromosomes are stained with two
fluorochromes, 4',6-diamidino- 2-phenylindole (DAPI) at
final concentration of 1.5 µM and mithramycin A (MI) at
final concentration of 20 µM for at least 30 min. MgSO4
is added to chromosome suspension at final
concentration of 10 mM prior to staining.

13. METAPHASES OF FEULGEN-STAINED PEA
(PISUM SATIVUM) ROOT TIP CHROMOSOMES-S.
LUCRETTI, G. GUALBERTI, AND J. DOLEZEL
• A) and B): metaphases of Feulgen-stained
pea (Pisum sativum L.) root tip
chromosomes observed by their
fluorescent emission after green
excitation, Standard and reconstructed
karyotype L-84, respectively.
• C) and D): flow-karyotyping histograms of
DAPI-stained chromosome suspensions
for the Standard and L-84, respectively.
Capital letters indicates chromosome
specific peaks, as assigned after
chromosome sorting.

14. FLOW-KARYOTYPING OF DNA INTEGRAL FLUORESCENCE
(FPA) OF DAPI-STAINED PEA CHROMOSOMES, S. LUCRETTI, G.
GUALBERTI, J. MACAS, AND J. DOLEZEL
• Flow-karyotyping of DNA integral
fluorescence (FPA) of DAPI-stained pea
chromosomes. Inside pictures show
sorted chromosomes from regions R1
(I+II) and R2 (VI+III and I), DAPI-
stained; from regions R3 (III+IV) and
R4 (V+VII) after PRINS labelling for
rDNA (chromosomes IV and VII with
secondary constriction are labelled)

15. MAPPING OF EUKARYOTIC &
PROKARYOTIC GENES

16. MAPPING OF PROKARYOTIC GENES
• Gene information is processed differently in prokaryotes & eukaryotes.
Because prokaryotes lack nuclear envelope, messenger RNA (mRNA) can
associate with ribosome in the cytoplasm as the mRNA is being formed.
• The following features are important for gene recognition:
(1) ORF length
(2) presence of a ribosome binding site (RBS) upstream of the start codon
(3) specific pattern of codon usage that is different from triplet frequencies in
non-coding regions (‘coding potential’), as well as other similar statistical
parameters; and
(4) similarity to known genes.

17.  Intrinsic approach to gene recognition
Intrinsic, or ab initio, approaches use the first three types of data. Hidden Markov models (HMM) provide a
convenient language for integrating these diverse parameters of candidate genes in genomic sequences.
Extrinsic methods rely on the comparative analysis of genomic DNA sequences using alignment with known
genes and proteins.
 Ribosome binding sites
Ribosome binding sites are located in the (20) ... (1) region upstream of start codons and serve to direct
ribosomes to the correct translation start position. A part of RBS is formed by the purine-rich Shine–
Dalgarno (SD) sequence, which is complementary to the 39 end of the 16S rRNA. A number of early papers
described methods for recognition of ribosome binding sites using statistical, pattern recognition or neural
network modelling of experimentally mapped sites.
There are two approaches to the recognition of ribosome binding sites in the absence of a learning sample
 One possibility is to rely on the universal mechanisms of RBS recognition via basepairing of the SD box
and the 39-terminus of the 16S rRNA
 The other possibility is to derive a ‘pseudo-learning’ sample of candidate translation initiation sites using
protein coding regions predicted by database search or statistical analysis.
A convenient technique for integration of diverse parameters is the HMM. HMM24 is a Markov chain of
hidden states. Each state is assigned a distribution of emission probabilities (Bernoulli or Markov) that
generate the observed nucleotide sequence.

18.  Extrinsic approaches
Extrinsic analysis involves sequence similarity searches. Candidate gene products are
searched against protein sequence databanks. BLASTX, the most popular program of
this class, performs six-frame translation of the query DNA and compares the
resulting amino acid sequences to known proteins.
The simplest way to combine the extrinsic and intrinsic approaches is to apply them in
parallel.
 MATERIALS AND METHODS
The prediction is done in three steps:
• Building the tables of orthologues.
• Applying a dynamic programming algorithm to align pairs of orthologous genes.
• Filtering of results and identification of suspicious gene starts and possible frame-
shifts.

19. MAPPING OF EUKARYOTIC GENES
• The genomes of eukaryotic organisms contain hundreds to thousands of genes (an
estimated 30,000-50,000 in humans). Yet there are only a handful of chromosomes.
Thus, each chromosome in a eukaryotic genome must contain a large number of
genes.
• The transmission of genes located on the same chromosome may violate Mendel’s
Law of Independent Assortment, particularly if they are located very close together
along the same arm of a chromosome.
• This set of lecture notes will explain why, and provide the theoretical basis for
mapping genes along a chromosome by following the degree to which they violate
Mendel’s Law of Independent Assortment during genetic crosses.

20.  Linkage and Crossing Over
• In eukaryotic species, each linear chromosome contains a long piece of DNA
• A typical chromosome contains many hundred or even a few thousand different genes
• The term “linkage” has two related meanings
• 1. Two or more genes can be located on the same chromosome
• 2. Genes that are close together tend to be transmitted as a unit
• Chromosomes are called linkage groups
• They contain a group of genes that are linked together
• The number of linkage groups is the number of types of chromosomes of the species
• For example, in humans
• 22 autosomal linkage groups
• An X chromosome linkage group
• A Y chromosome linkage group
• Genes that are far apart on the same chromosome may independently assort from each other due to crossing-over during
meiosis.
• Occurs during prophase I of meiosis
• Homologous chromosomes exchange DNA segments

21.  LINKAGE GROUPS
Chromosomes are called linkage groups
– They contain a group of genes that are linked together They contain a group of genes
that are linked together.
• The number of linkage groups is the number of types of chromosomes of the species
– For example, in humans
" 22 autosomal linkage groups “
An X chromosome linkage group "
A Y chromosome linkage group
• Genes that are far apart on the same chromosome can independently assort from
each other
• – This is due to This is due to crossing-over or recombination rossing-over or
recombination

22. PCR IN GENE MAPPING
• The polymerase chain reaction (PCR)’ is an in vitro method of nucleic acid synthesis that enables
the specific replication of a targeted segment of DNA, providing a rapid, highly sensitive, and
specific means of nucleic acid detection and isolation.
• The PCR technique uses two oligonucleotide primers that complement opposite ends of each strand
of a target sequence, and are oriented in such a way that DNA synthesis proceeds across the region
between the primers.
• PCR was originally performed using Klenow fragment as the DNA polymerase, and thermocycling
was accomplished by transferring reaction tubes between a series of water baths set at different
temperatures. The procedure required adding new enzyme after each cycle, and significant
manipulation of reaction tubes. PCR has since been improved through the use of thermostable Taq
DNA polymerase and automated instrumentation for thermal cycling. These ad- vances eliminated
the need to add fresh enzyme after each cycle, as well as the need for manual transfer of tubes
previously required to achieve heating and cooling. I

23. USES OF PCR IN GENE MAPPING
• Cloning a gene encoding a known protein: primers can be designed from the
sequence of amino acids or gene sequence. amplified product can be used as a probe
to pull out the full length gene from a cDNA or a genomic libraries.
• Amplification of old DNA: amplifying DNA sequence from museum material or
fossils to look at evolution of gene sequence( molecular evolution studies).
• Amplification of cloned DNA from vectors: convenient way of checking the inserts is
to amplify DNA & analysed it by southern blotting.
• Creating mutation in cloned DNA

24. USES OF PCR IN GENE MAPPING
• Rapid amplification of cDNA ends (RACE): most cloned in cDNA libraries are not of
full length. RACE enables 5’ or 3’ end of a transcript to be cloned as an alternative
to rescreening libraries for overlapping clones. Only one gene specific primer is
needed.
• Detection of bacterial & viral infection
• Detection of cancer : PCR technique is used for detecting mutation that occur in
cancer & monitoring cancer therapy
• Genetic diagnosis : PCR technique is also used in diagnosing inherited disorder like
cystic fibrosis, muscular dystrophy, haemophilia A & B & sickle cell anaemia.

25. MOLECULAR MAPS-RFLP, RAPD & THEIR
APLLICATION FOR DETECTION OF
ADULTERANTS
• Linkage maps of many plant species were limited in size until the advent of molecular mapping. The primary difficulty with
developing linkage maps was the inability to incorporate many markers into a single stock to be used for genetic analysis. This
inability occurred because of the deleterious effects of the expression of all mutant phenotypes in the single stock. Because normal
DNA or protein molecules are used to score the genetic material, molecular markers are phenotypically neutral. This is a
significant advantage compared to traditional phenotypic markers.
• The three most common types of markers used today are RFLP, RAPD and isozymes. Of the three marker types, RFLPs have
been used the most extensively. RFLP markers have several advantages in comparison with the RAPD and isozyme markers:
1) they are codominant and unaffected by the environment;
2) any source DNA can be used for the analysis; and
3) many markers can be mapped in a population that is not stressed by the effects of phenotypic mutations.
The primary drawback to RAPD markers is that they are dominant and do not permit the scoring of heterozygous individuals. The
weakness of isozyme markers is that each of the proteins that are being scored may not be expressed in the same tissue and at the
same time in development. Therefore several samplings of the genetic population need to be made.

26. • RFLP - Restriction Fragment Length Polymorphism; a molecular marker based on
the differential hybridization of cloned DNA to DNA fragments in a sample of
restriction enzyme digested DNAs; the marker is specific to a single clone/restriction
enzyme combination.
• RAPD - Randomly Amplified Polymorphic DNA; a molecular marker based on the
differential PCR amplification of a sample of DNAs from short oligonucleotide
sequences.
• AFLP - Amplified Fragment Length Polymorphism; a molecular marker generated
by a combination of restriction digestion and PCR amplification.
• Isozyme - a molecular marker system based on the staining of proteins with
identical function, but different electrophoretic mobility.

27. RFLP Loci
• RFLP analysis is an application of the Southern hybridization procedure. The general
principles will be explained here and we will then discuss several papers to obtain a
more in depth understanding of the procedure.
RAPD Loci
• RAPD markers have recently caught the fancy of many individuals in the field of applied
plant breeding. This molecular marker is based on the PCR amplification of random
locations in the genome of the plant. With this technique, a single oligonucleotide is used
to prime the amplification of genomic DNA. Because these primers are 10 nucleotides
long, they have the possibility of annealing at a number of locations in the genome. For
amplification products to occur, the binding must be to inverted repeats sequences
generally 150-4000 base pairs apart. The number of amplification products is directly
related to the number and orientation of the sequences that are complementary to the
primer in the genome

28. PHYSCAL MAPS IN-SITU HYBRIDIZATION
• In contrast to a linkage map, which specifies statistical distances between variable
DNA markers and genes in terms of recombination fractions (see “Classical Linkage
Mapping”), a physical map specifies physical distances between landmarks on the
DNA molecule of each chromosome.
1)Low-Resolution Physical Mapping by In-Situ Hybridization
2) High-Resolution Physical Mapping by Construction of
Contig Maps of Overlapping Clones

29. • The figure at right is a schematic of a
contig map for one chromosome. Right
now, the top priority of the Human
Genome Project is to construct a contig
map for each of the twenty-four
different chromosomes in the human
genome. Those maps, when integrated
with the corresponding genetic-linkage
maps, will provide a means of finding
the segments of DNA that contain
disease genes (see “Modern Linkage
Mapping”). The clones that make up
the map also provide the material
needed to sequence the human genome.

30. LOW-RESOLUTION PHYSICAL MAPPING BY IN-
SITU HYBRIDIZATION
• One standard low-resolution method for finding the physical position of a cloned fragment is in-situ
hybridization on metaphase chromosomes. We first find a segment within the cloned region whose base
sequence occurs nowhere else in the genome, We then synthesize many copies of a single strand of that
unique segment and label each copy with a fluorescent tag to make it useful as a DNA probe. A solution
containing the DNA probe is then applied to a spread of chromosomes that have been arrested at
metaphase and fixed to a microscope slide. (Metaphase is the phase of cell division during which
chromosomes have condensed to form the wormlike shapes easily visible under a light microscope.)
Under appropriate conditions the probe binds, or hybridizes, only to the chromosomal DNA with a base
sequence exactly complementary to that of the probe (see “Hybridization” in “Understanding
Inheritance”). The position on a metaphase chromosome where the probe has hybridized is imaged with a
fluorescence microscope as a bright spot. Because DNA molecules are wound very tightly during
metaphase, the resolution achieved with in-situ hybridization is low, about 3 million base pairs. In other
words, the hybridization signals from two probes less than 3 million base pairs apart will overlap one
another and cannot be resolved into two distinct spots. In-situ hybridization using four cloned inserts as
probes produced the bright spots on the metaphase chromosomes in the micrograph shown on the page
opposite.

31. HIGH-RESOLUTION PHYSICAL MAPPING BY
CONSTRUCTION OF CONTIG MAPS OF
OVERLAPPING CLONES
• To determine the positions of genomic landmarks with much greater resolution, we can replace the
chromosomes themselves with twenty-four contig maps, one for each of our twenty-two homologous
chromosome pairs and one for each of our two sex chromosomes. A contig map is a set of contiguous
overlapping cloned fragments that have been positioned relative to one another. In a complete
contig map for a human chromosome, the cloned fragments would include all the DNA present in
the chromosome and follow the same order found on the DNA molecule of the chromosome. As in
any physical map, distances are measured in base pairs. Using these contig maps, we can localize
any cloned fragment or other DNA probe, again by hybridization, to a much smaller portion of the
genome, namely to one of the cloned fragments in one of the maps. Moreover, we can determine the
position of any DNA probe relative to all other landmarks that have been similarly localized. Once
contig maps are constructed, the entire genome will be available as cloned fragments, and we will
be able to use these clones to analyze any region down to the level of its base sequence.

33. REFERENCES
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