Lecture - 2

Establishment of plant genome

mapping projects
I. Genome mapping
II. Use of molecular markers in Plant
Breeding
GENOME MAPPING

GENETIC MAPPING
PHYSICAL MAPPING
 GENOME MAPPING
Genetic mapping is based on the use of
genetic techniques to construct maps showing
the positions of genes and other sequence
features on a genome.
Genetic techniques include cross-breeding
experiments or,
Case of humans, the examination of family
histories (pedigrees).
Physical mapping uses molecular biology
techniques to examine DNA molecules
directly in order to construct maps showing
the positions of sequence features, including
genes.
 DNA MARKERS FOR GENETIC MAPPING
Mapped features that are not genes are called DNA
markers. As with gene markers, a DNA marker must have
at least two alleles to be useful. There are three types of
DNA sequence feature that satisfy this requirement:
Restriction fragment length polymorphisms (RFLPs)
Simple sequence length polymorphisms (SSLPs), and
i) Minisatellites, also known as variable number of
tandem repeats (VNTRs) in which the repeat
unit is up to 25 bp in length;
ii) Microsatellites or simple tandem repeats (STRs),
whose repeats are shorter, usually dinucleotide or
tetranucleotide units.
Single nucleotide polymorphisms (SNPs).
Restriction fragment length polymorphisms
(RFLP)
RFLP DETECTION
Restriction fragment length polymorphisms
(RFLPs)
Pedigree based on RFLP analysis
Linkage analysis shows that the disease gene D lies
between markers c and d.
 RFLP
Distance between RFLP markers is also
defined in recombination units or cM.
 Amplified Fragment Length Polymorphism
(AFLP)
AFLPs are differences in restriction fragment
lengths caused by SNPs or INDELs that create
or abolish restriction endonuclease recognition
sites.
The AFLP technique is based on the selective
PCR amplification of restriction fragments
from a total digest of genomic DNA
RAPD (Random Amplified Polymorphic DNA)

RAPD markers are DNA fragments from PCR

amplification of random segments of genomic
DNA with single primer of arbitrary
nucleotide sequence.
RAPD does not require any specific
knowledge of the DNA sequence of the target
organism
The identical 10-mer primers will or will not
amplify a segment of DNA, depending on
positions that are complementary to the
primers' sequence.
RAPD (Random Amplified
Polymorphic DNA)
 Simple sequence length polymorphisms (SSLPs),
Unlike RFLPs, SSLPs can be multi-allelic as each SSLP
can have a number of different length variants.
VNTRs - Minisatellites
VNTRs - Minisatellites
Microsatellites: simple tandem
repeats (STRs)
Simple tandem repeats (STRs)
 MAPPING TECHNIQUES
Linkage analysis is the basis of genetic mapping.
The offspring usually co-inherit either A with B or a
with b, and, in this case, the law of independent
assortment is not valid.
Thus to test for linkage between the genes for two
traits, certain types of matings are examined and
observe whether or not the pattern of the
combinations of traits exhibited by the offspring
follows the law of independent assortment.
If not, the gene pairs for those traits must be linked,
that is they must be on the same chromosome pair.
 What types of matings can reveal that the
genes for two traits are linked?
Only matings involving an individual who is
heterozygous for both traits (genotype AaBb) reveal
deviations from independent assortment and thus
reveal linkage.
Moreover, the most obvious deviations occur in the
test cross, a mating between a double heterozygote
and a doubly recessive homozygote (genotype
aabb).
Individuals with the genotype AaBb manifest both
dominant phenotypes; those with the genotype aabb
manifest both recessive phenotypes.
How do we estimate, from the offspring of a
single family, the likelihood that two gene
pairs are linked?

Recombination fraction
LOD score
Haldane mapping function
 Recombination Frequency
Recombination fraction is a measure of the distance
between two loci.
Two loci that show 1% recombination are defined as
being 1 centimorgan (cM) apart on a genetic map.
1 map unit = 1 cM (centimorgan)
Two genes that undergo independent assortment have
recombination frequency of 50 percent and are located on
nonhomologous chromosomes or far apart on the same
chromosome = unlinked
Genes with recombination frequencies less than 50
percent are on the same chromosome = linked
Calculation of Recombination Frequency

The percentage of recombinant progeny produced in a

cross is called the recombination frequency, which is
calculated as follows:
Recombination Frequency
Recombination fraction
 LOD SCORE
The LOD score is calculated as follows:
LOD = Z = Log10 probability of birth sequence with a given linkage
probability of birth sequence with no linkage

By convention, a LOD score greater than 2.5 is considered

evidence for linkage.

On the other hand, a LOD score less than 2.5 is considered

evidence to exclude linkage.
 LOD Score Analysis
The likelihood ratio is defined by :-
L(pedigree| = x)
L(pedigree| = 0.50)
where represents the recombination
fraction, where 0 x 0.49.
( (1 ) )
R NR

L.R. =
( 0.5) N

The LOD score (z) is the log10 (L.R.)

 Method to evaluate the statistical significance
of results.
Maximum-likelihood analysis, which estimates the most likely
value of the recombination fraction as well as the odds in favour
of linkage versus nonlinkage.
Given by Conditional probability L (data 1 ), which is the
likelihood of obtaining the data if the genes are linked and have a
recombination fraction of .
Likelihood of obtaining one recombinant and seven non-
recombinants when the recombination fraction is is proportional
to 1(1)7,
Where:
is, by definition, the probability of obtaining a recombinant ,
(1 ) is the probability of obtaining a nonrecombinant.
 Mapping function
The genetic distance between locus A and locus B is
defined as the average number of crossovers
occurring in the interval AB.
Mapping function is use to translate recombination
fractions into genetic distances.
In 1919 the British geneticist J. B. S. Haldane
proposed such Mapping function.
Haldane defined the genetic distance, x between two
loci as the average number of crossovers per meiosis
in the interval between the two loci.
 What is Haldane s mapping function ?
Assumptions: crossovers occurred at random along
the chromosome and that the probability of a
crossover at one position along the chromosome
was independent of the probability of a crossover at
another position.
Using these assumptions, he derived the following
relationship between
, the recombination fraction and
x , the genetic distance (in morgans):
= 1/2 (1-e-2x) or equivalently,
X= -1/2 ln (1-2)
 Genetic distance between two loci increases, the
recombination fraction approaches a limiting value
of 0.5.
Cytological observations of meiosis indicate that
the average number of crossovers undergone by
the chromosome pairs of a germ-line cell during
meiosis is 33.
Therefore, the average genetic length of a human
chromosome is about 1.4 morgans, or about 140
centimorgans.
Integration of MAP
 LIMITATIONS
A map generated by genetic techniques is rarely sufficient
for directing the sequencing phase of a genome project.
This is for two reasons:
The resolution of a genetic map depends on the number of
crossovers that have been scored .
Genes that are several tens of kb apart may appear at
the same position on the genetic map.
Genetic maps have limited accuracy .
Presence of recombination hotspots means that
crossovers are more likely to occur at some points
rather than at others.
Physical mapping techniques has been developed to
address this problem.
PHYSICAL MAPPING
 Physical mapping
Actual physical distances
Units in base-pairs
Contigs of large DNA fragments
Large insert DNA libraries (BACs, PACs, etc)
Restriction fragment fingerprinting
Minimum tiling set to cover entire genome
Correlation of genetic and physical maps
Genetic marker screening
EST screening
BAC-end sequencing
FISH
 Physical Mapping Systems
(like a Filing system of clones)
Yeast Artificial Chromosomes (YACs) 200-1000 kb

Bacteriophage P1 90 kb

Cosmids 40 kb

Bacteriophage l 9-23 kb

Plasmids (2-6 kb)

 Contigs
Contigs are groups of overlapping pieces of chromosomal
DNA
Make contiguous clones
For sequencing one wants to create minimum tiling path
Contig of smallest number of inserts that covers a region of
the chromosome

genomic DNA

contig

minimum
tiling path
 PHYSICAL MAPPING
Restriction mapping, which locates the
relative positions on a DNA molecule of the
recognition sequences for restriction
endonucleases;
Fluorescent in situ hybridization (FISH), in
which marker locations are mapped by
hybridizing a probe containing the marker to
intact chromosomes;
Sequence tagged site (STS) mapping, in
which the positions of short sequences are
mapped by PCR and/or hybridization analysis
of genome fragments.
The basic methodology for restriction
mapping
 Restriction mapping
partial restriction
 Physical maps
Physical maps can be generated by aligning the
restriction maps of specific pieces of cloned genomic
DNA (for instance, in YAC or BAC vectors) along the
chromosomes.
These maps are extremely useful for the purpose of map-
based gene cloning.
 Fluorescent in situ hybridization (FISH)

FISH enables the position of a marker on a

chromosome or extended DNA molecule to
be directly visualized
In FISH, the marker is a DNA sequence
that is visualized by hybridization with a
fluorescent probe.
In situ hybridization intact chromosome is
examined by probing it with a labeled DNA
molecule.
 In situ hybridization with radioactive or
fluorescent probes
The position on the chromosome at which hybridization
occurs provides information about the map location of the
DNA sequence used as the probe

DNA in the chromosome is made single stranded

(denatured).

The standard method for denaturing chromosomal DNA

without destroying the morphology of the chromosome is
to dry the preparation onto a glass microscope slide and
then treat with formamide.
Can distinguish chromosomes by painting using DNA
hybridization + fluorescent probes during mitosis
FISH
 FISH

16
16

DNA appears as a yellow band on chromosome16, thus locating this

particular simple sequence to one site in the genome.
 Sequence tagged site (STS) mapping
A sequence tagged site or STS is simply a
short DNA sequence, generally between
100 and 500 bp in length, that is easily
recognizable and occurs only once in the
chromosome or genome being studied.
To map a set of STSs, a collection of
overlapping DNA fragments from a single
chromosome or from the entire genome is
needed.
STS mapping
 STS mapping
The data from which the map will be derived
are obtained by determining which fragments
contain which STSs.
The chances of two STSs being present on
the same fragment will, of course, depend on
how close together they are in the genome.
The data can therefore be used to calculate
the distance between two markers.
Each map distance is based on the frequency
at which breaks occur between two markers
 Genetic V/s Physical
Distance
Map distances based on recombination
frequencies are not a direct measurement of
physical distance along a chromosome
Recombination hot spots overestimate
physical length
Low rates in heterochromatin and centromeres
underestimate actual physical length
Genetic vs. Physical Distance
Genetic and physical maps may differ in relative distances
and even in the position of genes on a chromosome.
 Map-based sequencing
The first method for assembling short,
sequenced fragments into a whole-genome
sequence, called a map-based approach,
Requires the initial creation of detailed
genetic and physical maps of the genome,
It provide known locations of genetic
markers (restriction sites, other genes, or
known DNA sequences) at regularly
spaced intervals along each chromosome.
Map-based sequencing
 Molecular marker applications in plant breeding
Molecular markers are used in plant breeding, taxonomy, physiology,
embryology, genetics, evolution, genetic engineering etc
Some of applications in plant breeding are:

1. Marker-assisted selection
It replaces evaluation of a trait (difficult or expensive).
It is not influenced by growth stage and environmental factors.
It can increase efficiency of selection for low-heritability.

2. Genetic diversity analysis

Plant breeding is dependant of genetic variation
Genetic diversity is measured by genetic distance (GD) or genetic
similarity (GS = 1 - GD)
It is used to assess variation over time, protection of intellectual
property rights, registration of germplasm, and evaluation of new
sources of germplasm for their potential to increase genetic diversity
It is superior to morphological, pedigree, heterosis, and biochemical
Cont...
...Cont
3. Genetic mapping
Markers can be used for map construction.
It predicts the linear arrangement of markers on a chromosome
and the recombination frequency.
4. Manipulating traits controlled by a few major loci
To map and transfer the gene of interest
5. Authentication or DNA fingerprinting
For diagnosis, protection property right and quality controlling
6. Phylogeny and evolution
Perform phylogenic analysis on a species by comparing the
presence/absence of various markers in their genome.
For taxonomic classification to determine the primary, secondary
or tertiary genepool of the system
 Summary
Plant breeding depends on genetic variation and selection.
Selection is difficult and assisted by morphological, biochemical
and molecular markers.
Molecular markers are DNA marker, polymorphic, independent of
environmental factors, easy, fast, co-dominant and reproducible
but it is costly.
The two forms of molecular techniques (hybridization and PCR
methods).
Many forms of molecular markers
RFLP, RAPD, VNTR, SSR, AFLP .
Molecular markers are used in diversity analysis, parent selection,
germplasm characterization, identification, genetic fingerprinting,
genetic diagnostics, genome organization and phylogenic analysis.