1. Methods for characterization of
animal genomes (RFLP, RAPD,
SNP, STR, QTL )
Submitted by:
Dr. Vijayata

2. Restriction Fragment Length Polymorphism
(RFLP)
• RFLP is a technique in which organisms may be differentiated
by analysis of patterns derived from cleavage of their DNA.
• If two organisms differ in the distance between sites of
cleavage of particular Restriction Endonucleases, the length of
the fragments produced will differ when the DNA is digested
with a restriction enzyme.
Potential of Molecular Markers in Plant Biotechnology, P. Kumar1&2, V.K. Gupta2, A.K. Misra2, D. R. Modi*1 and B.
K. Pandey2 Plant Omics Journal 2(4):141-162 (2009) ISSN: 1836-3644

3. • The similarity of the patterns generated can be used to
differentiate species (and even strains) from one another.
• This technique is mainly based on the special class of enzyme
i.e. Restriction Endonucleases.
• They have their origin in the DNA rearrangements that occur
due to evolutionary processes, point mutations within the
restriction enzyme recognition site sequences, insertions or
deletions within the fragments, and unequal crossing over
(Schlotterer & Tautz, 1992).
Potential of Molecular Markers in Plant Biotechnology, P. Kumar1&2, V.K. Gupta2, A.K. Misra2, D. R. Modi*1 and B.
K. Pandey2 Plant Omics Journal 2(4):141-162 (2009) ISSN: 1836-3644

4. • This is not always the case with genomic DNA molecules
because some restriction sites are polymorphic, existing as two
alleles, one allele displaying the correct sequence for the
restriction site and therefore being cut when the DNA is treated
with the enzyme, and the second allele having a sequence
alteration so the restriction site is no longer recognized.
• The result of the sequence alteration is that the two adjacent
restriction fragments remain linked together after treatment with
the enzyme, leading to a length polymorphism .
Genomes, 2nd edition Terence A BROWN Chapter 5. Mapping Genomes, 5.2.2. DNA markers for genetic mapping.
Oxford: Wiley-Liss; 2002.ISBN-10: 0-471-25046-5

5. The DNA molecule on the left has a polymorphic restriction site (marked with the
asterisk) that is not present in the molecule on the right. The RFLP is revealed
after treatment with the restriction enzyme because one of the molecules is cut
into four fragments whereas the other is cut into three fragments.
A restriction fragment length polymorphism (RFLP)
Genomes, 2nd edition Terence A BROWN Chapter 5. Mapping Genomes, 5.2.2. DNA markers for genetic mapping.
Oxford: Wiley-Liss; 2002.ISBN-10: 0-471-25046-5

6. • In order to score an RFLP, it is necessary to determine the size
of just one or two individual restriction fragments against a
background of many irrelevant fragments.
• Size fractionation is achieved by gel electrophoresis and, after
transfer to a membrane by Southern blotting; fragments of
interest are identified by hybridization with radioactive labeled
probe.
• Different sizes or lengths of restriction fragments are typically
produced when different individuals are tested.
Potential of Molecular Markers in Plant Biotechnology, P. Kumar1&2, V.K. Gupta2, A.K. Misra2, D. R. Modi*1 and B.
K. Pandey2 Plant Omics Journal 2(4):141-162 (2009) ISSN: 1836-3644

7. • Southern hybridization, using a probe that spans the
polymorphic restriction site, provides one way of visualizing
the RFLP , but nowadays PCR is more frequently used.
• The primers for the PCR are designed so that they anneal
either side of the polymorphic site, and the RFLP is typed by
treating the amplified fragment with the restriction enzyme
and then running a sample in an agarose gel .
Genomes, 2nd edition Terence A BROWN Chapter 5. Mapping Genomes, 5.2.2. DNA markers for genetic mapping.
Oxford: Wiley-Liss; 2002.ISBN-10: 0-471-25046-5

8. (A) RFLPs can be scored by Southern hybridization. The DNA is digested
with the appropriate restriction enzyme and separated in an agarose gel.
The smear of restriction fragments is transferred to a nylon membrane
and probed with a piece of DNA that spans the polymorphic restriction
site. If the site is absent then a single restriction fragment is detected
(lane 2); if the site is present then two fragments are detected (lane 3).
Two methods for scoring an RFLP :
Genomes, 2nd edition Terence A BROWN Chapter 5. Mapping Genomes, 5.2.2. DNA markers for genetic mapping.
Oxford: Wiley-Liss; 2002.ISBN-10: 0-471-25046-5

9. (B) The RFLP can also be typed by PCR, using primers that anneal either
side of the polymorphic restriction site. After the PCR, the products are
treated with the appropriate restriction enzyme and then analyzed by
agarose gel electrophoresis. If the site is absent then one band is seen on
the agarose gel; if the site is present then two bands are seen.
Genomes, 2nd edition Terence A BROWN Chapter 5. Mapping Genomes, 5.2.2. DNA markers for genetic mapping.
Oxford: Wiley-Liss; 2002.ISBN-10: 0-471-25046-5

10. Applications:
• RFLPs can be applied in diversity and phylogenetic studies
ranging from individuals within populations or species, to
closely related species.
• RFLPs have been widely used in gene mapping studies
because of their high genomic abundance due to the ample
availability of different restriction enzymes and random
distribution throughout the genome (Neale & Williams 1991).
• RFLP markers were used for the first time in the construction
of genetic maps by Botstein et al.1980.
Potential of Molecular Markers in Plant Biotechnology, P. Kumar1&2, V.K. Gupta2, A.K. Misra2, D. R. Modi*1 and B.
K. Pandey2 Plant Omics Journal 2(4):141-162 (2009) ISSN: 1836-3644

11. Random Amplified Polymorphic DNA
(RAPD)
• PCR based technology.
• RAPD is a DNA polymorphism assay based on the
amplification of random DNA segments with single primers of
arbitrary nucleotide sequence (Williams et al., 1990; Welsh and
McClelland, 1990).
Potential of Molecular Markers in Plant Biotechnology, P. Kumar1&2, V.K. Gupta2, A.K. Misra2, D. R. Modi*1 and B.
K. Pandey2 Plant Omics Journal 2(4):141-162 (2009) ISSN: 1836-3644

12. • In this reaction, a single species of primer anneals to the genomic
DNA at two different sites on complementary strands of DNA
template.
• If these priming sites are within an amplifiable range of each other, a
discrete DNA product is formed through thermo cyclic
amplification.
• On an average, each primer directs amplification of several discrete
loci in the genome, making the assay useful for efficient screening
of nucleotide sequence polymorphism between individuals .
Potential of Molecular Markers in Plant Biotechnology, P. Kumar1&2, V.K. Gupta2, A.K. Misra2, D. R. Modi*1 and B.
K. Pandey2 Plant Omics Journal 2(4):141-162 (2009) ISSN: 1836-3644

13. • Amplified products are separated on agarose gel (1.5-2.0%)
and visualised by ethidium bromide staining .
• The use of a single oligonucleotide promotes the generation of
several discrete DNA products and these are considered to
originate from different genetic loci.
• Polymorphisms result from mutations or rearrangements either
at or between the primer binding sites and are detected as the
presence or absence of a particular RAPD band .
• This means that RAPDs are dominant markers and, therefore,
can not be used to identify heterozygotes.
Random amplified polymorphic DNA (RAPD) markers and its applications
N. Senthil Kumar1 and G. Gurusubramanian2 , 2011 Sci Vis 11 (3), 116-124.

14. • The standard RAPD utilises short synthetic oligonucleotides
(10 bases long) of random sequences as primers to amplify
nanogram amounts of total genomic DNA under low annealing
temperatures by PCR.
• Welsh and McClelland independently developed a similar
methodology using primers about 15 nucleotides long and
different amplification and electrophoretic conditions from
RAPD and called it the arbitrarily primed polymerase chain
reaction (AP-PCR) technique.
Random amplified polymorphic DNA (RAPD) markers and its applications
N. Senthil Kumar1 and G. Gurusubramanian2 , 2011 Sci Vis 11 (3), 116-124.

15. • PCR amplification with primers shorter than 10 nucleotides
[DNA amplification finger- printing (DAF)] has also been
used to produce more complex DNA fingerprinting profiles.
• Although these approaches are different with respect to the
length of the random primers, amplification conditions and
visualization methods, they all differ from the standard PCR
condition in that only a single oligonucleotide of random
sequence is employed and no prior knowledge of the genome
subjected to analysis is required.
Random amplified polymorphic DNA (RAPD) markers and its applications
N. Senthil Kumar1 and G. Gurusubramanian2 , 2011 Sci Vis 11 (3), 116-124.

16. Applications:
• The application of RAPDs and their related modified markers
in variability analysis and individual-specific genotyping has
largely been carried out, but is less popular due to problems
such as poor reproducibility faint or fuzzy products, and
difficulty in scoring bands, which lead to inappropriate
inferences.
• RAPDs have been used for many purposes, ranging from
studies at the individual level (e.g. genetic identity) to studies
involving closely related species.
Potential of Molecular Markers in Plant Biotechnology, P. Kumar1&2, V.K. Gupta2, A.K. Misra2, D. R. Modi*1 and B.
K. Pandey2 Plant Omics Journal 2(4):141-162 (2009) ISSN: 1836-3644

17. • RAPDs have also been applied in gene mapping studies to fill
gaps not covered by other markers (Williams et al. 1990,
Hadrys et al. 1992).
• Variants of the RAPD technique include Arbitrarily Primed
Polymerase Chain Reaction (AP-PCR), which uses longer
arbitrary primers than RAPDs, and DNA Amplification
Fingerprinting (DAF) that uses shorter, 5–8 bp primers to
generate a larger number of fragments.
• Multiple Arbitrary Amplicon Profiling (MAAP) is the
collective term for techniques using single arbitrary primers.
Potential of Molecular Markers in Plant Biotechnology, P. Kumar1&2, V.K. Gupta2, A.K. Misra2, D. R. Modi*1 and B.
K. Pandey2 Plant Omics Journal 2(4):141-162 (2009) ISSN: 1836-3644

18. Microsatellites / Short tandem repeats (STRs)
• The term microsatellites was coined by Litt & Lutty (1989)and
it also known as Simple Sequence Repeats (SSRs).
• Microsatellites, or short tandem repeats/simple sequence
repeats (STRs/SSRs), are polymorphic loci present in DNA
that consist of repeating units of one to six base pairs in length
(CACACACACACACACA) (Litt and Lutty, 1989; Tautz,
1989).
A Brief Review of Molecular Techniques to Assess Plant Diversity,Arif et al.,2010
Int. J. Mol. Sci. 2010, 11, 2079-2096; doi:10.3390/ijms11052079

19. • The repeated sequence is often simple, consisting of two, three
or four nucleotides (di-, tri- and tetra- nucleotide repeats) and
can be repeated many times.
• Microsatellites can be amplified for identification by PCR
using the unique sequences of flanking regions as primers.
A Brief Review of Molecular Techniques to Assess Plant Diversity,Arif et al.,2010
Int. J. Mol. Sci. 2010, 11, 2079-2096; doi:10.3390/ijms11052079

20. • The most common way to detect microsatellites is to design
PCR primers that are unique to one locus in the genome and
that base pair on either side of the repeated portion.
• Therefore, a single pair of PCR primers will work for every
individual in the species and produce different sized products
for each of the different length of microsatellites.
• The PCR products are separated either by slab gel
electrophoresis or capillary gel electrophoresis in an automated
sequencer.
A Brief Review of Molecular Techniques to Assess Plant Diversity,Arif et al.,2010
Int. J. Mol. Sci. 2010, 11, 2079-2096; doi:10.3390/ijms11052079

21. • The number of repeats can be determined by carrying out a
PCR using primers that anneal either side of the STR, and then
examining the size of the resulting product by agarose or
polyacrylamide gel electrophoresis .
GENE CLONING AND DNAANALYSIS An Introduction T.A. BROWN, Faculty of Life Sciences University of
Manchester Manchester, Sixth Edition Part II The Applications of Gene Cloning and DNAAnalysis in Research P180

22. • Another approach using SSRs for the detection of polymorphism,
called inter simple sequence repeat (ISSR), involves PCR
amplification of genomic DNA between two microsatellite loci
using a single primer composed of a microsatellite sequence
anchored at the 3’ or 5’ end with one or few arbitrary, often
degenerate nucleotides.
• These primers target simple sequence repeats that are abundant
throughout the eukaryotic genome and do not require prior
knowledge of DNA sequence for primer design.
Biotechnology & Genetic Engineering Reviews Volume 25, Editor: Stephen E. Harding, First published 2008

23. • Microsatellites have proved to be versatile molecular markers,
particularly for population analysis, but they are not without
limitations.
• With the abundance of PCR technology, primers that flank
microsatellite loci are simple and quick to use, but the
development of correctly functioning primers is often a tedious
and costly process.
A Brief Review of Molecular Techniques to Assess Plant Diversity,Arif et al.,2010
Int. J. Mol. Sci. 2010, 11, 2079-2096; doi:10.3390/ijms11052079

24. Applications:
• In general, microsatellites show a high level of polymorphism.
• As a consequence, they are very informative markers that can be
used for many population genetics studies, ranging from the
individual level (e.g. clone and strain identification) to that of
closely related species (distinguish closely related genotypes;
because of their high degree of variability).
• Microsatellites are also considered ideal markers in gene
mapping studies .
Potential of Molecular Markers in Plant Biotechnology, P. Kumar1&2, V.K. Gupta2, A.K. Misra2, D. R. Modi*1 and B.
K. Pandey2 Plant Omics Journal 2(4):141-162 (2009) ISSN: 1836-3644

25. Single Nucleotide Polymorphism (SNP)
• Single nucleotide polymorphism (SNP) is a DNA sequence
variation occurring when a single nucleotide (A, T, G or C)
differs among members of a species. (Single nucleotide
variations in genome sequence of individuals of a population are
known as SNPs.)
A Brief Review of Molecular Techniques to Assess Plant Diversity,Arif et al.,2010

26. • They are bi-allelic markers, indicating a specific
polymorphism in two alleles only of a population.
• Most SNPs (about two of every three SNPs), involve the
replacement of cytosine (C) with thymine (T).
These are positions in a genome where some individuals have one nucleotide
(e.g. a G) and others have a different nucleotide (e.g. a C)
Genomes, 2nd edition Terence A BROWN Chapter 5. Mapping Genomes, 5.2.2. DNA markers for genetic mapping.
Oxford: Wiley-Liss; 2002.ISBN-10: 0-471-25046-5

27. • SNPs represent one of the more interesting approach in
genotypization, because they are abundant in the genome,
genetically stable and amenable to high-throughput automated
analysis (Vignal et al., 2002).
• SNP genotyping technologies have two components -
a method for determining the type of base present at a given
SNP locus , and a method for reporting the presence of the
allele(s) (signal detection).
MOLECULAR MARKERS IN ANIMAL GENOME ANALYSIS, A. Teneva
Biotechnology in Animal Husbandry 25 (5-6), p 1267-1284, 2009

28. • SNP detection is more rapid because it is based on
oligonucleotide hybridization analysis.
• An oligonucleotide is a short single-stranded DNA molecule,
usually less than 50 nucleotides in length, that is synthesized in
the test tube.
Genomes, 2nd edition Terence A BROWN Chapter 5. Mapping Genomes, 5.2.2. DNA markers for genetic mapping.
Oxford: Wiley-Liss; 2002.ISBN-10: 0-471-25046-5

29. • An oligonucleotide will hybridize with another DNA molecule
only if the oligonucleotide forms a completely base-paired
structure with the second molecule.
• If there is a single mismatch - a single position within the
oligonucleotide that does not form a base pair - then
hybridization does not occur .
Genomes, 2nd edition Terence A BROWN Chapter 5.
Mapping Genomes, 5.2.2. DNA markers for genetic
mapping. Oxford: Wiley-Liss; 2002.ISBN-10: 0-471-
25046-5

30. • Oligonucleotide hybridization can therefore discriminate
between the two alleles of an SNP.
• Various screening strategies have been devised ( Mir and
Southern, 2000), including DNA chip technology and
solution hybridization techniques.
• A DNA chip is a wafer of glass or silicon, 2.0 cm2 or less in
area, carrying many different oligonucleotides in a high-
density array.
• The DNA to be tested is labeled with a fluorescent marker and
pipetted onto the surface of the chip.
Genomes, 2nd edition Terence A BROWN Chapter 5. Mapping Genomes, 5.2.2. DNA markers for genetic mapping.
Oxford: Wiley-Liss; 2002.ISBN-10: 0-471-25046-5

31. • Hybridization is detected by examining the chip with a fluorescence
microscope, the positions at which the fluorescent signal is emitted
indicating which oligonucleotides have hybridized with the test
DNA.
• Many SNPs can therefore be scored in a single experiment (Gerhold
et al., 1999).
• Solution hybridization techniques are carried out in the wells of a
microtiter tray, each well containing a different oligonucleotide, and
use a detection system that can discriminate between unhybridized
single-stranded DNA and the double-stranded product that results
when an oligonucleotide hybridizes to the test DNA.
Genomes, 2nd edition Terence A BROWN Chapter 5. Mapping Genomes, 5.2.2. DNA markers for genetic mapping.
Oxford: Wiley-Liss; 2002.ISBN-10: 0-471-25046-5

32. • SNP have become the preferred markers in genetic disease
studies for various livestock species, as researches direct their
attention towards functional genomics (White et al., 2001).
• SNPs are becoming especially important as markers because they
are very stable, i.e. have very low mutation rates and can be
amplified with PCR for testing.
• One disadvantage of these markers is the lower informational
content compared with that of a highly polymorphic
microsatellite, but it can be compensated by the use of a higher
number of markers (Werner et al. 2002, 2004).
MOLECULAR MARKERS IN ANIMAL GENOME ANALYSIS, A. Teneva

33. Quantitative trait loci (QTLs)
• The regions within genomes that contain genes associated with a
particular quantitative trait are known as quantitative trait loci
(QTLs).
• The identification of QTLs based only on conventional phenotypic
evaluation is not possible.
• A major breakthrough in the characterization of quantitative traits
that created opportunities to select for QTLs was initiated by the
development of DNA (molecular) markers in the 1980s.
An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: The basic
concepts , B.C.Y. Collard1,4,∗, M.Z.Z. Jahufer2, J.B. Brouwer3 & E.C.K. Pang1 , 2005. Springer 142: 169–196.

34. QTL mapping
• The process of constructing linkage maps and conducting QTL
analysis to identify genomic regions associated with traits is known
as QTL mapping (also ‘genetic,’ ‘gene’ or ‘genome’ mapping) .
• The most important use for linkage maps is to identify chromosomal
locations containing genes and QTLs associated with traits of
interest; such maps may then be referred to as ‘QTL’ (or ‘genetic’)
maps.
• ‘QTL mapping’ is based on the principle that genes and markers
segregate via chromosome recombination (called crossing-over)
during meiosis (i.e. sexual reproduction), thus allowing their
analysis in the progeny (Paterson, 1996a).

35. • QTL analysis is based on the principle of detecting an association
between phenotype and the genotype of markers.
• Markers are used to partition the mapping population into different
genotypic groups based on the presence or absence of a particular
marker locus and to determine whether significant differences
exist between groups with respect to the trait being measured .
• A significant difference between phenotypic means of the groups
(either 2 or 3), depending on the marker system and type of
population, indicates that the marker locus being used to partition
the mapping population is linked to a QTL controlling the trait.
An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: The basic
concepts , B.C.Y. Collard1,4,∗, M.Z.Z. Jahufer2, J.B. Brouwer3 & E.C.K. Pang1 , 2005. Springer 142: 169–196.

36. • Three widely-used methods for detecting QTLs are single-
marker analysis, simple interval mapping and composite
interval mapping (Liu, 1998; Tanksley, 1993).
• Single-marker analysis (single-point analysis) is the
simplest method for detecting QTLs associated with single
markers.
• The statistical methods used for single-marker analysis include
t-tests, analysis of variance (ANOVA) and linear regression.
• This method does not require a complete linkage map and can
be performed with basic statistical software programs.
An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: The basic
concepts , B.C.Y. Collard1,4,∗, M.Z.Z. Jahufer2, J.B. Brouwer3 & E.C.K. Pang1 , 2005. Springer 142: 169–196.

37. • However, the major disadvantage with this method is that the
further a QTL is from a marker, the less likely it will be
detected.
• This is because recombination may occur between the marker
and the QTL.
• This causes the magnitude of the effect of a QTL to be
underestimated (Tanksley, 1993).
• The use of a large number of segregating DNA markers
covering the entire genome may minimize both problems
(Tanksley, 1993).
An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: The basic
concepts , B.C.Y. Collard1,4,∗, M.Z.Z. Jahufer2, J.B. Brouwer3 & E.C.K. Pang1 , 2005. Springer 142: 169–196.

38. • The simple interval mapping (SIM) method makes use of
linkage maps and analyses intervals between adjacent pairs of
linked markers along chromosomes simultaneously, instead of
analyzing single markers (Lander & Botstein, 1989).
• The use of linked markers for analysis compensates for
recombination between the markers and the QTL, and is
considered statistically more powerful compared to single-
point analysis (Lander & Botstein, 1989; Liu, 1998).
An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: The basic
concepts , B.C.Y. Collard1,4,∗, M.Z.Z. Jahufer2, J.B. Brouwer3 & E.C.K. Pang1 , 2005. Springer 142: 169–196.

39. • Composite interval mapping (CIM) has become popular for
mapping QTLs.
• This method combines interval mapping with linear regression
and includes additional genetic markers in the statistical model in
addition to an adjacent pair of linked markers for interval
mapping (Jansen, 1993; Jansen & Stam, 1994).
• The main advantage of CIM is that it is more precise and
effective at mapping QTLs compared to single-point analysis and
interval mapping, especially when linked QTLs are involved.
An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: The basic
concepts , B.C.Y. Collard1,4,∗, M.Z.Z. Jahufer2, J.B. Brouwer3 & E.C.K. Pang1 , 2005. Springer 142: 169–196.

40. Type of markers Advantages Disadvantages
Restriction Fragment
Length Polymorphism
(RFLP)
-High genomic abundance
-Co-dominant markers
-Highly reproducible
-Good genome coverage
-Can be used across species
-No sequence information
-Needed for map based cloning
-Need large amount of good
quality DNA
-Laborious (compared to RAPD)
-Difficult to automate
-Need radioactive labeling
-Cloning and characterization of
probe are required
Randomly Amplified
Polymorphic DNA
(RAPD)
-High genomic abundance
-Good genome coverage
-No sequence information
-Ideal for automation
-Less amount of DNA
-No radioactive labeling
-Relatively faster
-No probe or primer information
-Dominant markers
-Not reproducible
-Can not be used across species
-Not very well-tested
Simple Sequence
Repeat (SSR)
-High genomic abundance
-Highly reproducible
-Fairly good genome coverage
-High polymorphism
-No radioactive labeling
-Easy to automate
-Multiple alleles
-Can not be used across species
-Need sequence information
-Not well-tested

41. Feature RFLP RAPD STR/SSR
Amount of DNA
required
High Low
Low
Maximum
theoretical
number of possible
loci in analysis
Limited by the
restriction site
(nucleotide)
Polymorphism
Limited by the
size of genome, and
by nucleotide
polymorphism
Limited by the size of
genome and number of
simple repeats in a
Genome
Dominance Codominant Dominant Codominant
Genomic
abundance
High High Medium
Locus specificity Yes No No
Reproducibility
High to very
high
Low to
medium
Medium to
high
Type of
probes/primers
Low copy genomic
DNA or
cDNA clones
Usually 10 bp
random nucleotides
Specific repeat DNA
sequence
MOLECULAR MARKERS IN ANIMAL GENOME ANALYSIS, A. Teneva
Comparison of commonly used genetic markers (Mburu and Hanotte , 2005)

42. Feature RFLP RAPD STR/SSR
Degree of
Polymorphism
High Low Medium
Type of
polymorphism
Single base changes,
insertion, deletion
Single base changes,
insertion, deletion
Changes in length of
repeats
Potential for
studying
adaptive genetic
Variation
Limited Limited Limited
Transferability
Across related
species
Across genera Within species
Within genus or
species
Ease of development Difficult Easy Difficult
Technical
requirement
High Low Medium
Major application Physical mapping Gene tagging Genetic diversity
MOLECULAR MARKERS IN ANIMAL GENOME ANALYSIS, A. Teneva

43. Thank You