ABT302: Molecular Breeding

Molecular Markers- Introduction

Lecture-1
Date:22.10.2021
 Traits
 Genetic variation can be expressed in observable phenotypes-
Traits
 Traits are categorized into two groups
 Traits
 Qualitative traits:
1. Easily scored
2. Classified in the discrete classes
3. Complete correspondence between phenotype and genotypes
(a) Gregor Mendel carefully selected such contrasting traits for his
study. (b) Evaluated them for 2 years
 Some traits show continuous variation- quantitative traits
 Multigene
 influenced by environment
 They are not good indicator of genotypes
 Phenotypic selection is always dissappointing
 Markers
 Morphological Markers
 Biochemical Markers
 Molecular Markers

Properties of ideal markers

1. Polymorphic and multiallelic
2. Co-dominant
3. Should not be epistatic
4. Abundant and uniformly distributed
5. Should not be pleotropic
6. Should not influenced by environment
 Morphological Markers
 Easily scored by naked eye- naked eye polymorphism
 Not require sophisticated equipment or preparatory procedures
 scoring of these markers is simple, rapid, and inexpensive

Limitations:
 The number of good visible/morphological markers is limited.
 Generally, they can be scored only on whole plants and that too during
specific developmental stages.
 Many traits, e.g., disease resistance, may have a threshold requirement
for their expression.
 Some genes governing the marker traits may have pleiotropic
effect on the trait of interest,
 Finally, maintenance of suitable genetic stocks expressing the various
marker traits would be necessary.
 Biochemical Markers
 Protein-based markers are detected as electrophoretic variants of
proteins, including enzymes.
 These markers are generated by small changes in the coding sequences
leading to the alteration in the amino acid sequences
 The variant protein molecules differ from the wild-type molecules in
electrical charge detected as differential electrophoretic mobility
 Isozymes are different forms of an enzyme with same catalytic function
but different electrophoretic mobility in the same individual.
 isozymes are closely related variants of an enzyme encoded by different
genes, which may have arisen by gene duplication or polyploidization.
 In contrast, the variants of an enzyme encoded by different alleles of the
same gene are called allozymes.
 Therefore, only allozymes will behave as alleles of a marker locus and
will be useful in linkage analyses.
 Biochemical Markers
Advantages
 They reflect differences in gene sequences more directly than
visible/morphological markers
 only a small amount of tissue is needed for their detection
 they can often be detected at seedling stage or even from seeds
 analysis of one marker does not interfere with other protein-based
markers.
 Therefore, many different marker loci can be analyzed in the same cross.
 isozymes are usually codominant,
 their analysis is relatively easy
 data interpretation is facilitated by numerous reference data
 Biochemical Markers
Limitations
 Any two parents may be polymorphic for only a relatively small number
of protein-based markers.
 Isozymes represent only a small, non-random sample of the structural
genes of an organism.
 They detect only such mutations that produce a functional enzyme with
changed electrophoretic mobility.
 In addition, a single band may represent two different isozymes having
identical mobility.
 Finally, they may vary with the tissue, the developmental stage, and the
environment data interpretation is facilitated by numerous reference
data
 DNA Markers
 The DNA-based markers represent variation in genomic DNA sequences
of different individuals.

 They are detected as differential mobility of fragments in a gel,

hybridization with an array or PCR amplification, or as DNA sequence
differences.

 The development of these markers began in 1974, when analysis of

fragments generated by restriction enzyme digestion of DNA was used for
physical mapping of a gene in adenovirus.

 In 1980, human geneticists observed that digestion of genomic DNA with

restriction enzymes generated DNA fragments of different lengths from
the same genomic regions of different individuals.
 Ideal DNA Markers
 It should generate a very large number of polymorphic markers
 Evenly distributed throughout the genome.
 It should be codominant
 It should have multiple alleles to provide adequate resolution of genetic
differences among individuals/lines.
 The detection of marker alleles, i.e., genotyping, should be simple, easy,
quick, inexpensive, reproducible, and amenable to automation and have
high throughput.
 only small amount of DNA should be needed for genotyping,
 the error in genotyping should be near zero.
 Finally, the marker system should not require prior information about the
genome of an organism.
 However, none of the available marker systems meets all the above
criteria, but SNPs are the closest to being ideal molecular markers
 Advantage of DNA Markers
 Highly polymorphic markers
 Uniformly distributed over the entire genome.
 The number of different marker loci is very large
 DNA markers are generally neutral- No pleotropism
 Scoring for one DNA marker usually has no effect on that of the others
 Molecular markers show simple Mendelian inheritance.
 Marker genotyping is independent of the prevailing environment
 the marker assays are nondestructive. Therefore, MAS can be effectively
used under any environment and at any stage of development since the
trait phenotypes are not evaluated.
 MAS can also be used for an allele that is not expressed in the available
genotypes, e.g., a recessive allele in heterozygotes.
 The DNA samples can be stored for future use,
 specific marker stocks are not required.
 Application of DNA Markers
 fingerprinting of strains/varieties for unequivocal identification;
 mapping of genes and quantitative trait loci (QTLs);
 efficient MAS for tightly linked QTLs and such oligogenes, direct selection for
which may be costly or problematic;
 positional cloning of genes/QTLs;
 identification of chromosome segments that would contribute to improvements in
the target traits;
 establishing phylogenetic relationships among different strains/species;
 selection of parents for hybridization;
 assessing the basis of somaclonal variation;
 identification of pathogen races and biotypes;
 prediction of heterotic cross combinations;
 Identification of wide hybrids;
 gene pyramiding; and management and utilization of genetic resources.
 MAS allows the use of off-season nursery and greenhouse facilities to
reduce the time needed for variety development.
 Categories of DNA Markers
Based on chronology
 First generation (RFLP , RAPD)
 Second generation (SSR, AFLP and modification)
 Third generation (EST, ddRAD, GBS and SNPs)

Based on use of PCR

 Non-PCR based (RFLP)
 PCR based (RAPD, SSR, AFLP and modification)

Based on location and function

 Random (RAPD )
 Gene based (STS, genic-SSR)
 Functional markers

Based on ease of assay

 Low throughput
 Medium throughput
 High throughput
 Types of DNA Markers
 restriction fragment length polymorphism (RFLP),  (12) cleaved amplified polymorphic sequences (CAPSs),

 (2) randomly amplified polymorphic DNAs (RAPDs),  (13) allele-specific PCR,

 (3) arbitraryprimed PCR,  (14) allelespecific ligation,

 (4) DNA amplification fingerprinting (DAF),  (15) single-strand conformation polymorphism (SSCP),

 (5) amplified fragment length polymorphism (AFLP),  (16) diversity array technology (DArT),

 (6) sequence-characterized amplified regions (SCAR),  (17) inter-SSR (ISSR) markers,

 (7) sequence-tagged sites (STS),  (18) amplicon length polymorphism (ALP),

 (8) allele-specific associated primers (ASAP),  (19) sequence-related amplified polymorphism(SRAP),

 (9) single primer amplification reactions (SPARs),  (20) target region amplification polymorphism (TRAP),

 (10) simple sequence repeat (SSR) polymorphisms,  (21) transposable elementbased markers, and

 (11) SSR-anchored PCR,  (22) single-nucleotide polymorphism (SNP)

 RFLP
 Restriction fragment length polymorphism (RFLP) produces DNA
fragments of different length at particular locus in two or more
individuals when digested with restriction enzyme.
 Botstein et al. (1980) described the principle and the procedure of RFLP
Detection of polymorphism by RFLP
 Advantages of RFLP
a very large number of RFLP loci can be scored and mapped

RFLP marker does not require the associated gene to express itself;

they are highly reproducible,

are codominant in nature, and

allow mapping of even QTLs; and

construction of RFLP maps is very rapid

 Advantages of RFLP
a very large number of RFLP loci can be scored and mapped

RFLP marker does not require the associated gene to express itself;

they are highly reproducible,

are codominant in nature, and

allow mapping of even QTLs; and

construction of RFLP maps is very rapid

 Limitations of RFLP
The RFLP procedure is expensive and requires considerable labor and time.
 The original method used radioactive probes that are hazardous to handle
and require special disposal facilities.
 Considerable skill and effort is needed for the development of RFLPs,
including the construction of genomic/cDNA libraries for the identification of
suitable probes.
 The DNA used for RFLP analysis must be of high purity to enable restriction
digestion.
 Further, scoring of RFLPs in different individuals/lines takes far greater
time and effort than that for many other molecular markers like SSRs.
 Finally, this marker system is not amenable to automation and high-
throughput analysis.
Conversion of RFLP into PCR-based markers
The RFLP can be converted into user friendly PCR-based markers.

 This requires sequencing of two ends of useful RFLP locus

Primers are designed from the ends

This procedure is known as PCR-RFLP

If considerable length polymorphism detected, this marker can be

used as a STS (Sequence tagged site)

 STS shows polymorphism due to Insertion or deletion

If not this marker can be used as CAPS (Cleaved amplified

polymorphic sequence)
Conversion of RFLP into PCR-based markers
The RFLP can be converted into user friendly PCR-based markers.

 This requires sequencing of two ends of useful RFLP locus

Primers are designed from the ends

This procedure is known as PCR-RFLP

If considerable length polymorphism detected, this marker can be

used as a STS (Sequence tagged site)

 STS shows polymorphism due to Insertion or deletion

If not this marker can be used as CAPS (Cleaved amplified

polymorphic sequence)
ABT302: Molecular Breeding

Molecular Markers- RAPD

Lecture-2
Date:25.10.2021
 Quick revision
 What is qualitative traits?

 Differentiate between qualitative and quantitative traits?

 What are different types markers?

 Advantage and disadvantages of diff classes of markers?

 Who developed the RFLP marker?

 What is the basis of polymorphism in RFLP markers?

 What are the advantage of RFLP marker?

 How can an RFLP locus be converted into PCR-based marker?

 What are the disadvantage of RFLP marker?

Randomly Amplified Polymorphic DNA
 Developed by Williams et al in 1980

 Uses PCR to amplify the set of random primers without using

radioactive probes.

 Required a single, short (~10 nt) primer

 Same primers act as forward and reverse primers

 Primer designing does not require sequence information

 Random primers bind to template at several site

 410=1,048,576 combinations
PCR
PCR
Basis of polymorphism in RAPD
Basis of polymorphism in RAPD
RAPD Primers
 Advantages of RAPD
 No prior knowledge of sequence information is required

 High level of polymorphism

 Small amount of DNA is required

 Small random primers

 Less time consuming

 Very safe-No radioactive probes are required

 Suitable for genetic diversity assessment and DNA fingerprinting

 Disadvantages of RAPD
 Non reproducible

 Significant deviation in the Mendelian ratio

 Dominant marker

 Primers are random and non -specific

1. What will happen if the length of primer is increased ?

2. How can dominant RAPD can be converted to co-dominant

markers ?
Variation of RAPD marker

1. DNA amplification fingerprinting (DAF)

2. Arbitrary primed PCR (AP-PCR)

3. Sequence characterized amplified Region (SCAR)

 Variation of RAPD marker
1. DNA amplification fingerprinting (DAF)

 CaetanoAnolles et al. (1991) developed the procedure of DAF

 A single short primers of 4–6 nt are used

 ~100 bands are resulted

 resolved in PAGE

 Short extension times are sufficient for complete extension

 Scored as presence/absence

 Suitable for DNA fingerprinting

 Variation of RAPD marker
2. Arbitrary primed PCR (AP-PCR)

 Welsh and McClelland (1990) developed the procedure of AP-PCR

 Arbitrary sequence primers of 18–32 nt are used

 Amplification only occurs when some degree of complementarity

found at the 3’ end

 First two cycle performed at very low stringency and later at higher

 resolved in PAGE and ~100 bands can be expected for each ind.

 Scored as presence/absence

 Suitable for DNA fingerprinting

 Variation of RAPD marker
3. Sequence characterized amplified Region (SCAR)

 Developed by Paran and Michelmore (1993) from selected desirable

RAPD

 This term is also used for PCR-based markers derived from AFLP

 The desirable RAPD marker is eluted from the gel, cloned, and the

nucleotide sequences of its two termini are determined

 A pair of primers (usually, 20–24 not long), one forward and one

reverse primer, specific for the two terminal sequences is designed

 This primer pair is expected to amplify a single fragment and

More reliable marker

 SCAR polymorphisms are generally dominant (scored as “presence”

or “absence” of a single unique band)

 They can be used for physical as well as genetic mapping,

comparative mapping, and phylogenetic relationship studies.

Sequence Tagged Site (STS) Marker
 Developed by Olson et al 1989.

 A locus that can be unambiguously defined in terms of flanking

primer sequences that are used for its amplification is called

sequence-tagged site

 Required a pair of primers for an STS locus which amplifies a single

band
Sequence Tagged Site (STS) Marker
STSs can be created in the following four ways:

1. Specific bands of RAPD are eluted, cloned and sequenced. A

pair of PCR primers is designed from the ends. This strategy
generates SCAR markers.

2. The two ends of an RFLP or AFLP fragment are sequenced, and

specific primers are designed for amplification of the RFLP/AFLP
locus.

3. STSs are often created by determining the unique sequences

flanking mini- and microsatellite sites. A pair of primers specific
for these unique sequences is designed for PCR amplification of
each of these sites.

4. Sequences of ~400 bp long fragments of genomic DNA are

determined, ~20 bp primers are designed to amplify ~200–400 bp
segments. If a pair of primers amplifies a single product of the
correct size, a unique STS has been identified.
 CAPS Marker
The cleaved amplified polymorphic sequences (CAPSs) markers
are generated by restriction digestion of PCR amplified product.

CAPS are co-dominant marker

They result from alterations in the recognition sites, located
within the amplification products instead genomic DNA as in RFLP
Restriction enzyme should have 4 bp recognition since the
restriction sites are expected in the ~0.5–2 kb the amplification
products.

This technique is useful when the amplified DNA fragments are

large, fail to reveal polymorphism among genotypes, and contain
a SNP within the recognition site for a restriction enzyme.

The CAPS approach is preferable to the standard RFLP analyses.

But the use of restriction enzymes are costly, time consuming,

unsuitable for high-throughput analysis and automation.
CAPS Marker
 dCAPS Marker
The derived cleaved amplified
polymorphic sequences (dCAPSs)

It is the variant of CAPS marker

dCAPS differs from CAPS especially

in using specific primers, which are
designed with one or two
mismatches.

Amplified product is then digested

with suitable RE
ABT302: Molecular Breeding

Molecular Markers- AFLP

Lecture-3
Date:01.11.2021
 Quick revision
 Who developed the RAPD?

 What is the length of primers?

 What is the basis of polymorphism in RAPD?

 What are major disadvantages of RAPD ?

 What are major advantages of RAPD?

 What are variants of RAPD?

 How can a SCAR marker developed?

 What are the different methods of STS development?

 What is CAPS marker?

 How dCAPS are developed?

Amplified Fragment Length Polymorphism®
 AFLP is a trademark of KeyGene

 Developed by Zabeau and Vos (1993)

 It uses restriction enzymes of DNA followed by selective

amplification of fragments using PCR

 Dominant marker

 High level of polymorphism is detected

 Reproducible than RAPD

 Prior sequence information is not required

 Produces ~50-100 bands therefore suitable for high density

linkage maps
AFLP-Procedure
 Restriction Digestion
Isolation of genomic DNA

 Highly pure and good quality genomic DNA is isolated

Restriction digestion

 Genomic DNA is digested with restriction enzyme.

 It should be combination of rare cutter and frequent cutter eg.

Mse I 5’ TTAA 3’ Highly frequent

EcoRI or Pst I 5’ GAATTC 3’ or 5’ CTGCAG 3’ Less frequent

Mse I Mse I Pst I Pst I

I II III
 Adapter Ligation
 Selectively remove the highly frequent fragments

Adapter ligation

 Ligate the RE-specific adapter with the each ends of fragments

 Adapters has (1) a core sequence and (2) enzyme specific sequence
 Pre-amplification
Pre- amplification

 Pre- amplification is carried out a primer designed from core sequence

with 1 of 3 nucleotides at the 3’ end of primer

 This reduces a proportion of 1/16 = ¼ X ¼ per selective nucleotide

from the mixture for forward and reverse primer

 This method greatly reduce the proportion of mixture

 The primer should be radioactively labeled

 The amplification only favors Mse I –Pst I digested fragments

 The MseI-MseI and Pst I-Pst I will not amplify- i) primers in inverted

repeats , ii) amplified products form a hairpin

 Selective amplification
Selective amplification

 The amplified mixture is diluted to 10 fold

 This mixture acts as template for selective amplification PCR

 Carried out by PCR using adapter specific primers

 AFLP primer consists three parts : i) a core sequence, ii) enzyme

sequence, iii) selective nucleotides

Selective amplification
 Gel analysis
 Denaturing PAGE is used

 Amplicons are detected by autoradiograaphy

or fluorescentl

The procedure can be automated in a sequencer

 Disadvantage of AFLP
 Labor-intensive, costly, time consuming and technically demanding

 Use of restriction enzyme aid extra efforts

 Highly pure DNA preparation

 High background noise lead to complex data

 Use radioactive label primes

 Dominant but 4-15% loci are co-dominant that can detect homozygote

and heterozygote at few locus

 Advantage of AFLP
 High level of polymorphism

 Reproducible

 Does not require sequence information, therefore it can be

apply on non model organism

 identification, characterization of germplasm, high-resolution

mapping, marker-assisted selection (MAS), and gene cloning.

 Variants of AFLP
 Microsatellite AFLP or SAMPL (Selective Amplification of

Microsattelite Polymorphic DNA or sequence-tagged

microsatellite profiling (STMP)

ABT302: Molecular Breeding

SSRs and ISSRs

Lecture-4
Date:08.11.2021
 Quick revision
 Who developed the AFLP?

 Steps of development of AFLP marker?

 Components of adapters?

 Which of the following categories will be amplified

PstI-PstI

MseI-MseI

PstI-MseI
Quick revision

Nucleic Acids Res. 2001 Apr 15; 29(8): e44.

 Quick revision

 What is selective amplification?

 Disadvantages of AFLPs?

 Advantages of AFLPs?

 How can a AFLP markers converted to other markers?

 Repeat-based markers

DNA Sequence
Based on expression

• Coding Sequence

• Non-coding Sequence

(Regulatory Sequence)

Based on occurrence

• Unique Sequence

• Repetitive Sequence
 Repeat-based markers
Repetitive DNA Sequence
Repetitive Sequence

• Tandem Repeats

• Interspersed repeats

Micro-satellite markers (short , 2-6 nt, tandemly repeated sequence)

Mini-satellite markers (>10 nt or 10-100 nt tandem repeats )

 Microsatellite markers
 Developed by Litt and Luty (1989)

 Also known as Simple Sequence Repeats (SSRs) or Short tandem

repeats (STRs)

 They are short (2-6 nucleotides) tandem repeats, repeated

several times (100-1000 times) in the genome

 Co-dominant

 Multiallelic

 Relatively abundant in the genome

 Easy and less time consuming

 Can be automated
Microsatellite markers
Microsatellite markers
Microsatellite markers
 Discovery of SSRs

 SSR-enrichment method

 From Genome Sequence data

 SSR-enrichment method
SSR-enrichment from Genomic DNA library

 Isolate the high quality genomic DNA

 Fragmentation

 Cloning into suitable vectors

 Propagate the recombinant vectors into suitable host

 Design the probe containing repeats

 Hybridization of suitable of clone

 Sequencing and primer designing

 PCR amplification
Genomic DNA library enrichment
 Genome Sequencing data
Computational search of the genome sequencing data

 Retrieve the genome sequence

 Identify the repeats using softwares e.g. MISA, SSRIT etc

 Design primers

 PCR amplification

 Validation of SSR markers into suitable mapping population

General flow chart
Molecular Basis of Polymorphism in SSRs
Variation in SSR length is arises due to

 Unequal crossing over

 Replication slippage
 Stuttering of SSRs
 “Stutter bands” are the minor bands different from the main

amplification products
Inter Simple Sequence Repeats (ISSRs)
 They are also called ISA (inter SSR amplification).

 First developed by Zietkiewicz et al. 1994

 ISSR techniques is very much similar to the RAPD

 ISSR primers are designed from microsatellite regions

 They are 16–18-base pair long, 3’ anchored

 They are able to amplify the inter SSR sequences or

sequences flanking SSRs

 These regions are 100–3,000-bp long and flanked by closely
spaced, inversely oriented microsatellites

 Dominant markers
Inter Simple Sequence Repeats (ISSRs)
Inter Simple Sequence Repeats (ISSRs)
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