Application of Molecular Markers in Forensic Botany

Zagazig University
Faculty of Science
Microbiology and Botany
Department
Application of Molecular
markers in Forensic Botany
Presented By
Mostafa Ahmed Mahmoud Elkhashab

B.Sc Student, Biotechnology program,
Microbiology and Botany Department
Supervisor
Dr/ Ekram Mohamed

Lecturer of Molecular Genetics, Microbiology and Botany
Department
2019
Application of Molecular markers in Forensc Botany
Table of Contents
Table of Contents ................................................................................................. II
Table of figures ................................................................................................... III
Abstract ................................................................................................................. 1
Introduction ........................................................................................................... 2
FORENSIC SCIENCE ...................................................................................... 2
FORENSIC PLANT BIOTECHNOLOGY ....................................................... 3
Plant DNA Typing Methods ................................................................................. 4
1. Amplified Length Fragments Polymorphism (AFLP) .................................. 6
Basic steps of AFLP fingerprinting ................................................................ 8
Weaknesses of AFLP.................................................................................... 12
2. Random Amplified polymorphism DNA (RAPD) ...................................... 13
How It Works ............................................................................................... 15
Limitations of RAPD .................................................................................... 17
Can RAPD Analysis Help Catch a Killer? ......................................................... 19
Types of samples and collection for DNA analyses ........................................... 22
I. Leaves ........................................................................................................... 22
II. Flowers ........................................................................................................ 22
III. Wood .......................................................................................................... 22
IV. Seeds, fruits and roots .............................................................................. 23
Preservation of Samples................................................................................... 23
Experimental methodologies in NHFG .............................................................. 25
References ........................................................................................................... 27
II
Table of figures
Fig. 1. Lineology of DNA-based markers (Time Clock). .................................................... 4
Fig. 2. Illustration of the Polymerase Chain Reaction ...................................................... 7
Fig. 3. AFLP: Principle ...................................................................................................... 9
Fig. 4. DNA sequencer................................................................................................... 10
Fig. 5. AFLP electropherogram. ..................................................................................... 11
Fig. 6. Pictorial view methodology of RAPD. ................................................................. 15
Fig. 7. Strategy of PCR with arbitrary primers. .............................................................. 16
Fig. 8. A Comparison of Plant DNA Typing Methods ..................................................... 18
Fig. 9. Palo verde tree (Cercidium spp.) ........................................................................ 19
Fig. 10. Pipeline showing the main steps usually involved in processing forensic
nonhuman DNA samples.. ............................................................................................. 26
III
Abstract
Molecular markers based on DNA sequence have become a remarkable tool
in the Forensic Sciences for the identification of culprits. Now a day’s majority
of criminal cases are being solved based on DNA evidence from different
biological materials like blood, boon, semen, nails with skin piece, hair with hair
follicle, spores and any plant part etc. available at the scene of crime. Presently,
DNA evidence from plants have also played an important role in solving forensic
cases and DNA from any plant part found at the site of incidence can be used to
locate the murderers, kidnapers, victims or in arresting drug traffickers. All
molecular markers are not useful in Forensic Botany, only some molecular
markers are used for plant DNA evidence which includes DNA barcoding, RAPD
(Random Amplified Polymorphic DNA), RFLP (Restriction Fragment Length
Polymorphism), AFLP (Amplified Fragment Length Polymorphism), SNP
(Single Nucleotide Polymorphism) and Microsatellites, but the most widely used
molecular marker for plant evident is SSR (Simple Sequence Repeats) due to its
high reproducibility with great discrimination power and error free results from
small piece of evidence.
1
Introduction
Molecular techniques allowed us to trace the dynamics of speciation and to
determine the relatedness of species and the genetic diversity within populations
(Gillan et al. 1995). At the present time molecular markers based on
mitochondrial (mt) DNA are used in wildlife forensics because mt DNA have
high exponential rate and less recombination ratio as compare to nuclear DNA
(Bhaskar, Khan, and Goyal 2011). Mitochondrial DNA is also transfer from
parents to offspring in the haploid form and their nucleotide sequence is also
important in identification and reorganization of species (H. M. Hsieh et al.
2001). Another gene used in species detection, finding phylogenetic relationship
and forensic studies is Cytochrome b gene (H. Hsieh et al. 2005). There is also a
wide use of large ribosomal 16S rRNA gene for species detection and
phylogenetic tree formation (Stubbs et al. 2000). Molecular markers which are
usually used in nucleotide sequencing of plant DNA comprise RAPD (randomly
amplified polymorphic DNA), AFLP (amplified fragment length
polymorphisms) SSR (simple sequence repeats or microsatellites) (Wan and Fang
2003). On the base of Polymerase Chain Reaction (PCR) molecular markers are
divided into non-PCR based markers and PCR based markers (Omondi et al.
2016).
FORENSIC SCIENCE
The word Forensic originated from a Latin word ‘forensis’ imply “of or for
the forum”. So Forensic Science is explained as appliance of scientific techniques
for providing justice and fair dealing (Koopman et al. 2012). Forensic Science is
further divided into different fields of science: forensic biology, forensic
anthropology (deals with the study of human remains for forensic investigation),
forensic chemistry, forensic physics, forensic botany, forensic entomology,
forensic serology (using body fluids such as blood, semen for forensic analysis)
etc. (Chandra and Sharma 2014). So, Forensic science is a set of different
disciplines like pathology, anthropology, genetics, toxicology deontology and
chemistry which is use for official investigation systems (Edlund 2010).
2
FORENSIC PLANT BIOTECHNOLOGY

Plant Biotechnology is utilization of a range of biological treatments for
elucidation of different areas of plant sciences (Chawla 2002). In the field of
forensic science plant biotechnology techniques such as DNA fingerprinting,
DNA bar-coding and use of Molecular markers is done to reveal the truth or solve
the mystery of case by spotting and categorizing species and finding locality of a
focused plant (Shukla 2016). Plant DNA bar-coding has limited scope in contrast
to animal DNA bar coding because in animals mitochondrial, cytochrome c,
oxidase I can be used as universal barcode system. As there is no gene for bar-
coding in plants that’s why in this situations, molecular markers like Short
Tandem Repeats (STRs), Single Nucleotide Polymorphisms (SNPs), Simple
Sequence Repeats (SSRs), Variable Number Tandem Repeats (VNTRs) can be
used in Forensic Botany (Zaya and Ashley 2012). Consequently, the division
between human and nonhuman forensic genetics (HFG and NHFG, respectively)
is not just the result of an anthropocentric historical tradition; rather, it could be
derived from the different genomic architectures of the involved organisms (Prinz
2008).
The initial appliance of molecular technique of plant DNA evident analysis
was done in 1992 to solve a murder case by molecular analysis of Palo Verde
tree’s seed pods which ensure the presence of suspect at crime spot (Yoon 1993).
When a crime is conducted in forest or in semi urban area, bryophytes can be a
good source of evidence because bryophytes DNA typing have ability to remain
stable in fluctuating environment (Virtanen, Korpelainen, and Kostamo 2007).
3
Plant DNA Typing Methods

In recent years, molecular markers and especially DNA-based markers, have
been extensively used in many areas such as gene mapping and tagging
(Kliebenstein, Gershenzon, and Mitchell-olds 2001; Karp et al. 1998),
characterisation of sex (Flachowsky et al. 2001; Martinez et al. 1999), analysis of
genetic diversity (Erschadi et al. 2002; Palacios, Kresovich, and González-
Candelas 1999; Lerceteau and Szmidt 1999; Godt and Hamrick 1999) or genetic
relatedness (Mace, Gebhardt, and Lester 1999; Roa et al. 1997; BROOKFIELD
1992). In population genetics, protein-based markers (allozymes) were the first
markers developed and widely used (Godt and Hamrick 1999). DNA-based
methodologies are now the method of choice to differentiate closely related
organisms (Widen, Cronberg, and Widen 2013; Ouborg, Piquot, and Van
Groenendael 1999; Slatkin 1994). Moreover, the use of DNA-based markers
allows efficient comparisons because genetic differences are detectable at all
stages of development of the organism unlike allozymes which may show age
dependent changes.
Fig. 1. Lineology of DNA-based markers (Time Clock).
4
The most frequently used markers in population genetics are allozymes

(biochimical), RAPD (Random Amplified Polymorphic DNA) (Williams et al.
1990), RFLP (Restriction Fragment Length Polymorphism) (Botstein et al. 1980),
AFLP (Amplified Fragment Length Polymorphism) (Zabeau and P.Vos 1993),
minisatellite fingerprints, microsatellites and SSR (Single Sequence Repeats)
(Tautz and Renz 1984).
Methods have been optimized to extract DNA for forensic analysis. Efforts
have been dedicated to purify samples to remove phenolic substances,
polysaccharides, and humic acids, which are often co-extracted with the DNA
extracted from plants and can inhibit the PCR reaction.
5
1. Amplified Length Fragments Polymorphism

(AFLP)
Amplified Length Fragments Polymorphism is a recent DNA fingerprinting
technique developed by (Zabeau and P.Vos 1993; Vos et al. 1995; Caetano-
Anolles and Gresshoff 1997). This method is based on PCR amplification of
selected restriction fragments of a total digested genomic DNA. Once labelled,
amplified products are separated by electrophoresis. DNA fragments obtained
range from 60 to 500 base pairs.
To be visualised, DNA polymorphism, which is usually made of small DNA
fragments of few base pairs (up to 500), must be amplified. This amplification is
commonly done by Polymerase Chain Reaction (K. B. Mullis and Faloona 1987;
K. Mullis et al. 1992). The PCR method can amplify specific DNA fragments
through a precise priming of the polymerisation reaction occurring at each end of
the target DNA. This precise priming is done by short oligonucleotidic sequences
(Primers) able to anneal to the template DNA in the target zone. Primers are 18-
24 base pairs long, synthesised in laboratory and correspond to a complementary
DNA sequence designed in the flanking regions of the heavy strand of the target
DNA (Figure 2).
6
Fig. 2. Illustration of the Polymerase Chain Reaction
7
Basic steps of AFLP fingerprinting

1. DNA extraction
Clean and high molecular weight DNA is a prerequisite for AFLP. DNA
extraction is a critical first step in the experimental workflow of DNA Sequencing
and Fragment analysis. The overall quality, accuracy and length of the DNA
sequence read can be significantly affected by characteristics of the sample itself,
and the method chosen for nucleic acid extraction. Ideal methods will vary
depending on the source or tissue type, how it was obtained from its source, and
how the sample was handled or stored prior to extraction.
2. Restriction
Restriction fragments of the genomic DNA are produced by using two different
restriction enzymes: a frequent cutter (the four-base restriction enzyme MseI) and
a rare cutter (the six-base restriction enzyme EcoRI) (Figure 3). The frequent
cutter serves to generate small fragments, which amplify well and which have the
optimal size range for separation on a sequence gel, whereas the rare cutter limits
the number of fragments to be amplified.
3. Ligation of oligonucleotide adapters
Double-stranded adapters consist of a core sequence and an enzyme-specific

sequence (Figure 3). Therefore, adapters are specific for either the EcoRI site or
the MseI site. Usually restriction and ligation take place in a single reaction.
Ligation of the adapter to the restricted DNA alters the restriction site in order to
prevent a second restriction from taking place after ligation has occurred. The
core sequence of the adapters consists of a known DNA sequence of 20
nucleotides, which will be used later as primer in the PCR.
4. Pre-amplification
This step is a normal PCR where the adapters are used as primers. This first
PCR, called preamplification, allows a first selection of fragments by only
amplifying the DNA restriction fragments that have ligated an adapter to both
extremities. Additionally, to the adapter sequences, the primers used for the pre-
selective amplification have a supplementary base. This extra base enables
another first selection by amplifying ¼ of the fragments that have ligated an
adapter to both extremities.
8
Fig. 3. AFLP: Principle
9
5. Amplification
The aim of this step is to restrict the level of polymorphism and to label the
DNA. For this second amplification, we added three more nucleotides at the 3’
end of the primer sequence used for the preamplification (= adapters sequence +
3 nucleotides; (Figure 3). These two additional nucleotides make the
amplification more selective and will decrease the number of restriction
fragments amplified (polymorphism). Moreover, one of the primers (usually the
EcoRI primer) is labelled with a fluorescent dye, and will allow the visualisation
of DNA during the migration.
6. Electrophoresis
The PCR products are denaturated and run on acrylamide gel (DNA sequencer)
(Figure 4). A thin capillary containing a polymer replaces the usual acrylamide
gel. The electrophoresis conditions we used for fragments analysis can resolve
DNA fragments differing just by one base pair. Samples are loaded in a track, and
run one after the other through the capillary.
Fig. 4. DNA sequencer
10
All fragments are separated with regard to length, smaller fragments running
first. Once passing the laser, a dye attached to the primer is excited and emits a
fluorescent signal that is then collected by a computer. The results of fluorescence
are visualized on the computer as peaks, called Electropherograms (Figure 5).
Each peak corresponds to a band on a normal acrylamide gel. Amplified
fragments range from 30 to 400 base pairs.
Fig. 5. AFLP electropherogram.
11
Weaknesses of AFLP
• Inability to differentiate between mixed and single-source samples.
• Need to use different kits adapted to the size of the genome being
analyzed.
• Developing locus-specific markers from individual fragments can be
difficult.
12
2. Random Amplified polymorphism DNA (RAPD)

Random amplified polymorphic DNA (RAPD) markers were developed and
applied in plants (Welsh and Mcclelland 1990; Williams et al. 1990). RAPD is a
PCR-based technique used to obtain and compare genetic fingerprints or profiles
within a strain or species. Commonly used for gene mapping studies, the RAPD
technique involves the use of synthetic oligonucleotide primers of arbitrary
sequence that randomly bind at complementary sequences along the plant
genome.
Despite enormous advances in molecular marker techniques that have been
used to obtain genetic data, RAPD remains a useful approach for several reasons
(Williams et al. 1990; Lacerda et al. 2002; Magalhães, Martinez, and Gaiotto
2007; Brahmane, Mitra, and Mishra 2008; Dutra et al. 2008; Soares et al. 2008).
The method has considerable advantages over other kinds of DNA marker
analysis because it is fast, requires little amount of DNA, is suitable for work on
anonymous genomes (HADRYS, BALICK, and SCHIERWATER 1992), is
economic, does not involve complex procedures of DNA sequencing, requires
only a simple laboratory with minimum equipment to perform PCR, and can
detect polymorphism in any kind of sequences.
In RAPD, DNA fingerprints that incorporate hundreds of polymorphic markers
can be produced relatively easily with little or no prior information about the
genetics of the organism (Bagley, Anderson, and May 2001). RAPD
fingerprinting is straightforward and can be optimized for reproducibility. The
versatility of this technique stems from the simplicity of the information provided
by the fingerprint, the high throughput of the method, and the ease with which
fingerprints can be generated. RAPD fingerprinting is a PCR-based technique
which utilizes a single, arbitrarily chosen short oligonucleotide primer to amplify
genome segments flanked by two complimentary primer binding sites in inverted
orientation (Williams et al. 1990).
Typically, in standard RAPD analysis, each primer is a 10-mer
oligonucleotide with 50–70% G +C content and with no self-complementary
ends. Short primers with an arbitrary sequence can be complimentary to a number
of sites in a genome. If the sites occur on opposite strands of segment of DNA in
inverted orientation and the distance between the sites are short (<5 kbp) enough
for PCR, then the segment flanked by the sites can be amplified (see examples
illustrated in Figs. 1 and 2). Amplifications on different segments are
13
independent. The primers will hybridize to binding site that are identical or highly
homologous to their template nucleotide sequences, although some mismatches
especially at the 5' end are allowed.
Genotyping technology: Total DNA is amplified using short single (10
nucleotides) random primers in PCR and the resulting fragments are size
separated on an ethidium bromide stained agarose gel.
Genotyping: The banding pattern is transformed onto a 1/0 matrix to
determine the existence or absence of each band.
Source of polymorphism: Homology between the primer and the
template DNA results in the existence of a certain band. Any mutation that
prevents the hybridization of a primer to the template DNA at a certain
locus results in the absence of this band.
The number of RAPD loci used for the analyses of plant genomes varies
between several tens to several hundreds. (Note: although the number of short,
random, 10 base primers is high, most of them are not polymorphic). RAPD are
multilocus markers and their mode of inheritance is dominant. The genotyping
technology is very simple (the major advantage of this system), therefore, RAPD
has become very popular in many laboratories.
The quality and quantity of amplified product are dependent on the following:
• The length of the primers (primer length will eventually determine the
complexity of the profile and the difficulty of analysis)
• The distance between each forward and reverse primer that allows for
efficient amplification (optimally <1,500 bp)
• The presence of sufficient variation within the primer-binding regions
to effectively differentiate between closely related samples
14
How It Works
In RAPD technology (Fig. 6), random short synthetic oligonucleotide primers
(10-12 base pairs) are used to amplify the genomic DNA through polymerase
chain reaction under low annealing temperature. The amplicons generated are
separated on agarose gels based on sizes. As stated that primer size is short,
therefore annealing temperature range is 28-38ºC. At this temperature range,
primers anneal wherever they find complementary sequences from the genome
and the profile of amplified DNA vary in size that depends on nucleotide
sequence homology and the primer at the end of each amplified product.
Fig. 6. Pictorial view methodology of RAPD.
Unlike traditional PCR analysis, 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. For example, no fragment is
produced if primers annealed too far apart or 3' ends of the primers are not facing
each other. Therefore, if a mutation has occurred in the template DNA at the site
that was previously complementary to the primer, a PCR product will not be
produced, resulting in a different pattern of amplified DNA segments on the gel.
15
RAPD polymorphisms can theoretically result from several types of events:

(1) insertion of a large piece of DNA between the primer binding sites may
exceed the capacity of PCR, resulting in fragment loss.
(2) insertion or deletion of a small piece of DNA will lead to a change in size
of the amplified fragment.
(3) the deletion of one of the two primer annealing sites results in either the
loss of a fragment or an increase in size.
(4) a nucleotide substitution within one or both primer target sites may affect
the annealing process, which can lead to a presence versus absence polymorphism
or to a change in fragment size (Figure 7).
Fig. 7. Strategy of PCR with arbitrary primers. Genomic DNA, a thermostable DNA
polymerase, one (or two) primer(s) of arbitrary sequence, the four deoxyribonucleotide
triphosphates (dNTPs) and a suitable buffer are combined into a reaction tube and subjected to
PCR. The primers anneal to anonymous target sequences of the template DNA. If two primers
(depicted as arrows, not drawn to scale) anneal in an opposite direction and at a suitable distance
from each other, the DNA sequence between the two primers is amplified. PCR products are
separated by gel electrophoresis and visualized by, e.g., ethidium bromide staining. Various
mechanisms may result in presence versus absence polymorphisms. For example, a base
substitution within a primer target site (indicated by x) may interfere with primer annealing, and
thus prevent the amplification of the respective fragment.
16
Limitations of RAPD
• Nearly all RAPD markers are dominant, i.e. it is not possible to
distinguish whether a DNA segment is amplified from a locus that is
heterozygous (1 copy) or homozygous (2 copies). Co-dominant RAPD
markers, observed as different-sized DNA segments amplified from the
same locus, are detected only rarely.
17
Fig. 8. A Comparison of Plant DNA Typing Methods
18
Can RAPD Analysis Help Catch a Killer?

In early May of 1992, the body of a deceased black woman identified as Denise
Johnson was discovered in Phoenix, AZ. Her nude, dead body was discovered
under a Palo Verde tree (Cercidium spp.) (Figure 9) and the tire tracks leading
from the scene were obvious to investigators. In addition, a pager was found near
the woman’s body, a number of large gouge marks were evident on the paloverde
tree, and scrape marks in the dirt demonstrated signs of an apparent struggle
(Rudin and Inman 2010). An eyewitness soon came forward to notify authorities
that a white pickup truck had been seen speeding away from the same area the
night before.
Fig. 9. Palo verde tree (Cercidium spp.)
19
A short time later, sheriff ’s deputies identified the owner of the pager as Mark
Bogan, who coincidentally owned a white pickup that had obvious damage to the
rear end. Bogan immediately became the primary suspect. With the pager as
damaging evidence, Bogan willfully admitted to picking up the hitchhiking
Johnson and having sexual intercourse with her. However, he vehemently denied
having anything to do with her homicide. He claimed that after having sex, the
two began to argue, and when he demanded that she get out of his truck, she
reached up onto the dashboard and stole a number of items from him, including
his pager (Attorney et al. 2001). Bogan assured the investigating officers that he
had never been in the same area as where the victim’s body had been found.
Since Bogan had admitted to picking up the victim, the idea of linking Denise
Johnson to the suspect’s pickup was irrelevant. The investigators would have to
link Bogan to the area in which the victim’s body had been found. With Bogan’s
statement in mind, detectives searched the suspect’s pickup for any evidence that
could connect him to the crime scene — and what lay in the truck’s bed was
exactly what they needed. Detectives found pods from a plant or tree that they
could not identify until returning to the scene where the victim was found. Back
at the crime scene, they noticed that the paloverde trees that surrounded the area
possessed the same pods that were recovered from the suspect’s pickup.
Theorizing that the pods found within the bed of Bogan’s truck may have come
from one of the gouged trees, the detectives questioned whether it was possible
to link the two pieces of evidence through genetic DNA comparison.
Following an extensive search for a genetic researcher who could accurately
perform the necessary testing, Judge Susan Bolton assigned the complicated task
to Dr. Timothy Helentjaris, a molecular geneticist from the University of Arizona.
Using RAPD technology, Helentjaris not only profiled the pods collected from
Bogan’s truck, but also created a database comprised of the trees located nearest
to the victim and others around the crime scene as well. This small database, along
with the fact that the paloverde tree population contains multiple genetic
variations, allowed Helentjaris to conclude that the pods located within Bogan’s
truck bed most closely matched that from the tree nearest the body (Yoon 1993).
20
However, since RAPD plant analysis had never been admitted into a criminal
trial, a Frye hearing was necessary. At the hearing, Northern Arizona University
professor Dr. Paul Keim testified on behalf of the defense and challenged the
reliability and validity of not only the plant RAPD method but the sample
collection and database creation as well. Dr. Keim insisted that in order to
accurately determine whether the pods found in the suspect’s truck were truly
from a tree found at the crime scene, a much larger database was needed for
statistical significance. Subsequently, Judge Bolton ruled that the evidence
submitted by Dr. Helentjaris could be heard during the trial, but due to Dr. Keim’s
database challenges, no statistical random match probabilities could be used.
With the aid of plant RAPD testing, good police work, and an abundance of other
forensic evidence, Bogan was sentenced to life in prison, which was upheld by
an appellate court in 1995 (Rudin and Inman 2010).
21
Types of samples and collection for DNA

analyses
A wide variety of plant tissues can be used for genetic analysis. Forensic
investigations should collect all samples that may be of use and determine their
suitability later. A sample not collected because its relevance is overlooked is lost
forever. Very little in the way of special tools or storage is needed, and most
samples can be inexpensively preserved for long-term storage and later analysis
should they become relevant to the investigation. However, the DNA in an
improperly preserved specimen can quickly become degraded and its forensic
value greatly limited. Proper collection and subsequent storage of samples will
increase their utility and prolong the window for successful genetic analysis.
I. Leaves
Leaves are generally the preferred tissue for genetic analysis and are what most
botanists use in routine studies. If collecting from live plants, one or two healthy,
green leaves (depending on size, giving a total of about 2 cm2) should be chosen.
Avoid leaves with evidence of rot, infection, or insects. If the evidence is not from
a living plant, or leaves are not available, other tissues can be of use. Dried or
dead leaves can be used, however DNA will degrade over time and the success
rates of these samples may be quite low (Craft, Owens, and Ashley 2007).
II. Flowers
Flowers, either living or dried, are also suitable samples and can provide good
quality DNA for analyses.
III. Wood
While decaying wood is unlikely to yield suitable DNA, there have been
several recent advances in the use of wood for genetic analysis (Finkeldey,
Leinemann, and Gailing 2010). DNA yields from wood samples tend to be lower
than from leaf tissue and compounds that inhibit polymerase chain reaction (PCR)
are often a problem, but methods are improving.
22
IV. Seeds, fruits and roots

Seeds (Walters et al. 2006) and fruits, and even roots (Kumar, Pushpangadan,
and Mehrotra 2003) can provide suitable DNA for genetic analyses. In short,
almost any plant tissue has potential, although the age, condition, and original
DNA content of the tissue will determine its utility. When in doubt, collect the
sample.
Preservation of Samples
One of the simplest methods of preserving plant samples for genetic analysis
is drying the sample in silica gel. Removing moisture from the sample inhibits
the enzymes that would otherwise start to break down the DNA and other cellular
components. Dried samples are also less susceptible to fungal growth. Leaves can
be placed in a sealed plastic bag (such as a small zipper bag) and a few grams of
silica gel added directly to the bag. Mixing some indicating silica gel will help
determine if the silica gel is exhausted and needs replacing. Once the leaves are
dry, the sample is stable at room temperature almost indefinitely.
Silica gel storage is applicable to most samples. While a dried leaf or wood
sample may be able to survive prolonged storage without silica gel, the addition
of silica gel may prolong the length of time DNA can be successfully extracted
and the sample will not deteriorate. Samples with exceptionally high moisture
content, such as cacti or succulents, may need to have their silica gel replaced
several times to achieve sufficient drying.
Room temperature storage without proper desiccation should be avoided if at
all possible. DNA is, however, a remarkably resilient molecule and it should
never be assumed that a sample will not yield DNA – DNA has been successfully
extracted from 150-year-old dried specimens (Rogers and Bendich 1985;
Savolainen et al. 1995), seeds from archeological sites (Cappellini et al. 2010),
and even a 17–20 million-year-old fossil (Soltis, Soltis, and Smiley 1992).
Experimentation with similarly preserved non-evidence samples become more
important with marginal samples, but if a reasonable genetic test can assist an
investigation, sample quality alone should not discourage attempts to conduct a
genetic analysis.
23
If silica gel desiccation is not practical, samples that will be analysed within a
few days can be stored at 4ºC (refrigerated). Freezing at -20ºC is suitable for
samples that need to be stored for a few months, and -80ºC, ultracold storage will
preserve samples for several years. In all cases, samples should be stored in
airtight plastic bags or containers. While these methods are feasible for small
quantities of samples, freezer space is typically far more limited and costly than
room temperature storage, thus desiccation as described above is generally
preferred.
24
Experimental methodologies in NHFG

The techniques of forensic genetics originally developed for humans were
rapidly adapted to other sources of genetic material. The experimental pipeline
used in NHFG (Fig 10) starts with a request for a genetic testing. Next, samples
are collected using a sampling kit (either commercial or assembled in the
laboratory) and transported to the laboratory under proper conditions.
An accurate description of the biological nature of the sample is usually
included, and a unique code must be assigned to each collected sample. If the
request is part of a legal procedure, not only traceability but also the strict
maintenance of the chain of custody (chronological documentation of the
evidence) are key issues. The procedure continues in the laboratory, where the
genetic material is extracted from the samples using an appropriate and validated
protocol.
Certain urgent situations (e.g., bioterrorism) may require the use of methods
that were not previously validated. The laboratory may have to deal with new
kinds of biological material or taxonomic groups never studied before. In such
cases, the laboratory has to be able to develop a valid strategy to extract DNA
with sufficient quality and quantity for downstream analyses.
25
Fig. 10. Pipeline showing the main steps usually involved in processing
forensic nonhuman DNA samples. The exact procedure will depend on the
conditions available at each laboratory. The process starts with the evaluation of
the case and sample collection (green boxes). The procedure continues in the
laboratory, where the DNA is extracted from the biological source material and
analyzed according to an appropriate protocol (blue boxes). The genetic
information is then compared with reference databases and the results are
described in a written report (red boxes).
26
References
Attorney, Staff, Two Reckless, Dog Owners, and Douglas Beamish. 2001.
“Nonhuman DNA Testing Increases DNA’s Power to Identify and Convict
Criminals” 6 (1): 1999–2002.
Bagley, M. J., S. L. Anderson, and B. May. 2001. “Choice of Methodology for
Assessing Genetic Impacts of Environmental Stressors: Polymorphism and
Reproducibility of RAPD and AFLP Fingerprints.” Ecotoxicology 10 (4):
239–44. https://doi.org/10.1023/A:1016625612603.
Bhaskar, Ranjana, Imran Khan, and Surendra Prasad Goyal. 2011. Identification
of Forensic Case Using Molecular Markers: A Case Study of Hyaena
(Hyaena Hyaena). International Journal of Pharma and Bio Sciences. Vol.
2.
Botstein, D, R L White, M Skolnick, and R W Davis. 1980. “Construction of a
Genetic Linkage Map in Man Using Restriction Fragment Length
Polymorphisms.” American Journal of Human Genetics 32 (3): 314–31.
http://www.ncbi.nlm.nih.gov/pubmed/6247908%0Ahttp://www.pubmedcen
tral.nih.gov/articlerender.fcgi?artid=PMC1686077.
Brahmane, Manoj P, Krishna Mitra, and Sudhanshu S Mishra. 2008. “RAPD
Fingerprinting of the Ornamental Fish Badis Badis (Hamilton 1822) and
Dario Dario (Kullander and Britz 2002) (Perciformes, Badidae) from West
Bengal, India .” Genetics and Molecular Biology . scielo .
BROOKFIELD, J F Y. 1992. “DNA Fingerprinting in Clonal Organisms.”
Molecular Ecology 1 (1): 21–26. https://doi.org/10.1111/j.1365-
294X.1992.tb00151.x.
Caetano-Anolles, Gustavo, and Peter M. Gresshoff. 1997. “AFLP Analysis.” In
DNA Markers : Protocols, Applications, and Overviews, 115–31. Wiley-
VCH. https://www.wiley.com/en-
us/DNA+Markers%3A+Protocols%2C+Applications%2C+and+Overviews
-p-9780471160670.
Cappellini, Enrico, M. Thomas P. Gilbert, Filippo Geuna, Girolamo Fiorentino,
Allan Hall, Jane Thomas-Oates, Peter D. Ashton, et al. 2010. “A
Multidisciplinary Study of Archaeological Grape Seeds.”
Naturwissenschaften 97 (2): 205–17. https://doi.org/10.1007/s00114-009-
0629-3.
Chandra, R, and V Sharma. 2014. “Forensic Botany: An Emerging Discipline of
Plant Sciences.” Indian Botanists Blog-o-Journal.
http://www.indianbotanists.com/2014/03/forensic-botany-emerging-
27
discipline-of.html.
Chawla, H. S. 2002. Introduction to Plant Biotechnology. Science Publishers.
Craft, Kathleen J., Jeffrey D. Owens, and Mary V. Ashley. 2007. “Application
of Plant DNA Markers in Forensic Botany: Genetic Comparison of Quercus
Evidence Leaves to Crime Scene Trees Using Microsatellites.” Forensic
Science International 165 (1): 64–70.
https://doi.org/10.1016/j.forsciint.2006.03.002.
Dutra, N C L, M P C Telles, D L Dutra, and N J Silva Junior. 2008. “Genetic
Diversity in Populations of the Viper Bothrops Moojeni Hoge, 1966 in
Central Brazil Using RAPD Markers.” Genetics and Molecular Research :
GMR 7 (3): 603–13.
Edlund, Hanna. 2010. “Sensitive Identification Tools in Forensic DNA
Analysis.” Digital Comperhansive Summaries of Uppsala Dissertations
from the Faculty of Medicine. Department of Genetics and Pathology,
Faculty of Medicine, Disciplinary Domain of Medicine and Pharmacy,
Uppsala University: Acta Universitatis Upsaliensis. http://uu.diva-
portal.org/smash/get/diva2:356815/FULLTEXT01.pdf.
Erschadi, S., G. Haberer, R. A. Torres-Ruiz, and M. Schöniger. 2002.
“Estimating Genetic Diversity of Arabidopsis Thaliana Ecotypes with
Amplified Fragment Length Polymorphisms (AFLP).” TAG Theoretical
and Applied Genetics 100 (3–4): 633–40.
https://doi.org/10.1007/s001229900099.
Finkeldey, Reiner, Ludger Leinemann, and Oliver Gailing. 2010. “Molecular
Genetic Tools to Infer the Origin of Forest Plants and Wood.” Applied
Microbiology and Biotechnology 85 (5): 1251–58.
https://doi.org/10.1007/s00253-009-2328-6.
Flachowsky, H, E Schumann, W E Weber, and A Peil. 2001. “Application of
AFLP for the Detection of Sex-Specific Markers in Hemp.” Plant Breeding
120 (4): 305–9. https://doi.org/10.1046/j.1439-0523.2001.00620.x.
Gillan, R., M. D. Cole, A. Linacre, J. W. Thorpe, and N. D. Watson. 1995.
“Comparison of Cannabis Sativa by Random Amplification of Polymorphic
DNA (RAPD) and HPLC of Cannabinoids: A Preliminary Study.” Science
and Justice - Journal of the Forensic Science Society 35 (3): 169–77.
https://doi.org/10.1016/S1355-0306(95)72658-2.
Godt, M J W, and J L Hamrick. 1999. “Population Genetic Analysis of
Elliottiaracemosa (Ericaceae), a Rare Georgia Shrub.” Molecular Ecology 8
(1): 75–82. https://doi.org/10.1046/j.1365-294X.1999.00539.x.
HADRYS, H, M BALICK, and B SCHIERWATER. 1992. “Applications of
28
Random Amplified Polymorphic DNA (RAPD) in Molecular Ecology.”

Molecular Ecology 1 (1): 55–63. https://doi.org/10.1111/j.1365-
294X.1992.tb00155.x.
Hsieh, Hsing-mei, Chin-cheng Tsai, Li-chin Tsai, Hsiao-ling Chiang, Rocky
Tai-ping Shih, Adrian Linacre, and James Chun-i Lee. 2005. Species
Identification of Meat Products Using the Cytochrome b Gene. Forensic
Science Journal. Vol. 4.
Hsieh, Hsing Mei, Hsiao Ling Chiang, Li Chin Tsai, Shu Ya Lai, Nu En Huang,
Adrian Linacre, and James Chun I Lee. 2001. “Cytochrome b Gene for
Species Identification of the Conservation Animals.” Forensic Science
International 122 (1): 7–18. https://doi.org/10.1016/S0379-0738(01)00403-
0.
Karp, A, K Edwards, G Caetano-Anollés, and M Peter Gresshoff. 1998. “DNA
Markers: A Global Overview.” In DNA Markers: Protocols, Aplications
and Overviews, 1–13. Wiley-VCH. https://www.wiley.com/en-
us/DNA+Markers%3A+Protocols%2C+Applications%2C+and+Overviews
-p-9780471160670.
Kliebenstein, Daniel J, Jonathan Gershenzon, and Thomas Mitchell-olds. 2001.
“Comparative Quantitative Trait Loci Mapping of Aliphatic, Indolic and
Benzylic Glucosinolate Production In.” Genetics 159 (1): 359 LP – 370.
http://www.genetics.org/content/159/1/359.abstract.
Koopman, Wim J.M., Irene Kuiper, Dick J.A. Klein-Geltink, Gerda J.H.
Sabatino, and Marinus J.M. Smulders. 2012. “Botanical DNA Evidence in
Criminal Cases: Knotgrass (Polygonum Aviculare L.) as a Model Species.”
Forensic Science International: Genetics 6 (3): 366–74.
https://doi.org/10.1016/j.fsigen.2011.07.013.
Kumar, Anil, P. Pushpangadan, and S. Mehrotra. 2003. “Extraction of High-
Molecular-Weight DNA from Dry Root Tissue OfBerberis Lycium Suitable
for RAPD.” Plant Molecular Biology Reporter 21 (3): 309–309.
https://doi.org/10.1007/bf02772807.
Lacerda, Daniela R, Maria D P Acedo, José P. de L. F. Filho, and Maria B
Lovato. 2002. “A Técnica de RAPD : Uma Ferramenta Molecular Em
Estudos de Conservação de Plantas.” Revista Brasileira de Plantas
Medicinais 3 (2): 87–92. http://dx.doi.org/10.1590/1983-084X/13_071.
Lerceteau, Estelle, and Alfred E Szmidt. 1999. “Properties of AFLP Markers in
Inheritance and Genetic Diversity Studies of Pinus Sylvestris L.” Heredity
82 (3): 252–60. https://doi.org/10.1046/j.1365-2540.1999.00472.x.
Mace, E. S., C. G. Gebhardt, and R. N. Lester. 1999. “AFLP Analysis of
29
Genetic Relationships in the Tribe Datureae (Solanaceae).” Theoretical and

Applied Genetics 99 (3–4): 634–41.
https://doi.org/10.1007/s001220051278.
Magalhães, Marcelo, Romari Alejandra Martinez, and Fernanda Amato Gaiotto.
2007. “Diversidade Genética de Litopenaeus Vannamei Cultivado Na Bahia
.” Pesquisa Agropecuária Brasileira . scielo .
Martinez, E A, C Destombe, M C Quillet, and M Valero. 1999. “Identification
of Random Amplified Polymorphic DNA (RAPD) Markers Highly Linked
to Sex Determination in the Red Alga Gracilaria Gracilis.” Molecular
Ecology 8 (9): 1533–38. https://doi.org/10.1046/j.1365-294x.1999.00721.x.
Mullis, K, F Faloona, S Scharf, R Saiki, G Horn, and H Erlich. 1992. “Specific
Enzymatic Amplification of DNA in Vitro: The Polymerase Chain
Reaction. 1986.” Biotechnology (Reading, Mass.) 24: 17–27.
http://www.ncbi.nlm.nih.gov/pubmed/1422010.
Mullis, Kary B., and Fred A. Faloona. 1987. “Specific Synthesis of DNA in
Vitro via a Polymerase-Catalyzed Chain Reaction.” Methods in Enzymology
155 (C): 335–50. https://doi.org/10.1016/0076-6879(87)55023-6.
Omondi, Emmanuel O., Thomas Debener, Marcus Linde, Mary Abukutsa-
Onyango, Fekadu F. Dinssa, and Traud Winkelmann. 2016. Molecular
Markers for Genetic Diversity Studies in African Leafy Vegetables.
Advances in Bioscience and Biotechnology. Vol. 07.
https://doi.org/10.4236/abb.2016.73017.
Ouborg, N J, Y Piquot, and J M Van Groenendael. 1999. “Population Genetics,
Molecular Markers and the Study of Dispersal in Plants.” Journal of
Ecology 87 (4): 551–68. https://doi.org/10.1046/j.1365-2745.1999.00389.x.
Palacios, CARMEN, STEPHEN Kresovich, and FERNANDO González-
Candelas. 1999. “A Population Genetic Study of the Endangered Plant
Species Limonium Dufourii (Plumbaginaceae) Based on Amplified
Fragment Length Polymorphism (AFLP).” Molecular Ecology 8 (4): 645–
57. https://doi.org/10.1046/j.1365-294X.1999.t01-1-00597.x.
Prinz, Mechthild. 2008. Review of: Nonhuman DNA Typing, Theory and
Casework Applications . Journal of Forensic Sciences. Vol. 53. CRC
Press/Taylor & Francis. https://doi.org/10.1111/j.1556-4029.2008.00838.x.
Roa, A. C., M. M. Maya, M. C. Duque, J. Tohme, A. C. Allem, and M. W.
Bonierbale. 1997. “AFLP Analysis of Relationships among Cassava and
Other Manihot Species.” Theoretical and Applied Genetics 95 (5–6): 741–
50. https://doi.org/10.1007/s001220050620.
Rogers, Scott O., and Arnold J. Bendich. 1985. “Extraction of DNA from
30
Milligram Amounts of Fresh, Herbarium and Mummified Plant Tissues.”

Plant Molecular Biology 5 (2): 69–76.
https://doi.org/10.1007/BF00020088.
Rudin, Norah, and Keith Inman. 2010. An Introduction to Forensic DNA
Analysis, Second Edition. An Introduction to Forensic DNA Analysis,
Second Edition. CRC Press. https://doi.org/10.1201/9781420058505.
Savolainen, Vincent, Philippe Cuénoud, Rodolphe Spichiger, Maria D.P.
Martinez, Michèle Crèvecoeur, and Jean François Manen. 1995. “The Use
of Herbarium Specimens in DNA Phylogenetics: Evaluation and
Improvement.” Plant Systematics and Evolution 197 (1–4): 87–98.
https://doi.org/10.1007/BF00984634.
Shukla, Ritesh K. 2016. “International Journal of Forensic Sciences Forensic
Biotechnology: Application of Flow Cytometry in Legal Medicine.”
Forensic Biotechnology: Application of Flow Cytometry in Legal Medicine
Int J Forens Sci. https://medwinpublishers.com/IJFSC/IJFSC16000103.pdf.
Slatkin, Montgomery. 1994. “Avise, J. C. (1993); Molecular Markers, Natural
History and Evolution. Chapman and Hall. Price £75.00. ISBN: 0-412-
03771-8(Hb). ISBN: 0-412-03981(Pb).” Journal of Evolutionary Biology 7
(6): 766–67. https://doi.org/10.1046/j.1420-9101.1994.7060766.x.
Soares, Thannya Nascimento, Lázaro José Chaves, Mariana Pires De Campos
Telles, José Alexandre Felizola Diniz-Filho, and Lucileide Vilela Resende.
2008. Landscape Conservation Genetics of Dipteryx Alata (“baru” Tree:
Fabaceae) from Cerrado Region of Central Brazil. Genetica. Vol. 132.
https://doi.org/10.1007/s10709-007-9144-7.
Soltis, P S, D E Soltis, and C J Smiley. 1992. “An RbcL Sequence from a
Miocene Taxodium (Bald Cypress).” Proceedings of the National Academy
of Sciences of the United States of America 89 (1): 449–51.
https://doi.org/10.1073/pnas.89.1.449.
Stubbs, S. L J, J. S. Brazier, P. R. Talbot, and B. I. Duerden. 2000. “PCR-
Restriction Fragment Length Polymorphism Analysis for Identification of
Bacteroides Spp. and Characterization of Nitroimidazole Resistance
Genes.” Journal of Clinical Microbiology 38 (9): 3209–13.
http://jcm.asm.org/content/38/9/3209.abstract.
Tautz, Diethard, and Manfred Renz. 1984. “Simple Sequences Are Ubiquitous
Repetitive Components of Eukaryotic Genomes.” Nucleic Acids Research
12 (10): 4127–38. https://doi.org/10.1093/nar/12.10.4127.
Virtanen, Viivi, Helena Korpelainen, and Kirsi Kostamo. 2007. “Forensic
Botany: Usability of Bryophyte Material in Forensic Studies.” Forensic
31
Science International 172 (2–3): 161–63.

https://doi.org/10.1016/j.forsciint.2006.11.012.
Vos, Pieter, Rene Hogers, Marjo Bleeker, Martin Reijans, Theo Van De Lee,
Miranda Hornes, Adrie Friters, et al. 1995. “AFLP: A New Technique for
DNA Fingerprinting.” Nucleic Acids Research 23 (21): 4407–14.
https://doi.org/10.1093/nar/23.21.4407.
Walters, Christina, Ann A. Reilley, Patrick A. Reeves, Jennifer Baszczak, and
Christopher M. Richards. 2006. “The Utility of Aged Seeds in DNA
Banks.” Seed Science Research 16 (3): 169–78.
https://doi.org/10.1079/ssr2006246.
Wan, Qiu Hong, and Sheng G. Fang. 2003. “Application of Species-Specific
Polymerase Chain Reaction in the Forensic Identification of Tiger Species.”
Forensic Science International 131 (1): 75–78.
https://doi.org/10.1016/S0379-0738(02)00398-5.
Welsh, John, and Michael Mcclelland. 1990. “Fingerprinting Genomes Using
PCR with Arbitrary Primers.” Nucleic Acids Research 18 (24): 7213–18.
https://doi.org/10.1093/nar/18.24.7213.
Widen, Bjorn, Nils Cronberg, and Marie Widen. 2013. “Genotypic Diversity,
Molecular Markers and Spatial Distribution of Genets in Clonal Plants, a
Literature Survey.” Folia Geobotanica & Phytotaxonomica 29 (2): 245–63.
Williams, John G.K., Anne R. Kubelik, Kenneth J. Livak, J. Antoni Rafalski,
and Scott V. Tingey. 1990. “DNA Polymorphisms Amplified by Arbitrary
Primers Are Useful as Genetic Markers.” Nucleic Acids Research 18 (22):
6531–35. https://doi.org/10.1093/nar/18.22.6531.
Yoon, C K. 1993. “Forensic Science. Botanical Witness for the Prosecution.”
Science 260 (5110): 894 LP – 895.
https://doi.org/10.1126/science.8493521.
Zabeau, M., and P.Vos. 1993. Selective restriction fragment amplification. A
general method for DNA fingerprinting. 0 534 858, issued September 24,
1993. https://patents.google.com/patent/EP0534858B2.
Zaya, David N., and Mary V. Ashley. 2012. “Plant Genetics for Forensic
Applications.” In Methods in Molecular Biology, edited by Nikolaus J
Sucher, James R Hennell, and Maria C Carles, 862:35–52. Totowa, NJ:
Humana Press. https://doi.org/10.1007/978-1-61779-609-8_4.
32

Application of Molecular Markers in Forensic Botany

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Application of Molecular Markers in Forensic Botany

Uploaded by

Copyright:

Available Formats

Zagazig University

Mostafa Ahmed Mahmoud Elkhashab

Dr/ Ekram Mohamed

Fig. 2. Illustration of the Polymerase Chain Reaction ...................................................... 7

Fig. 3. AFLP: Principle ...................................................................................................... 9

Fig. 4. DNA sequencer................................................................................................... 10

Fig. 5. AFLP electropherogram. ..................................................................................... 11

Fig. 6. Pictorial view methodology of RAPD. ................................................................. 15

Fig. 7. Strategy of PCR with arbitrary primers. .............................................................. 16

Fig. 8. A Comparison of Plant DNA Typing Methods ..................................................... 18

Fig. 9. Palo verde tree (Cercidium spp.) ........................................................................ 19

nonhuman DNA samples.. ............................................................................................. 26

FORENSIC PLANT BIOTECHNOLOGY

Plant DNA Typing Methods

Fig. 1. Lineology of DNA-based markers (Time Clock).

The most frequently used markers in population genetics are allozymes

1. Amplified Length Fragments Polymorphism

Fig. 2. Illustration of the Polymerase Chain Reaction

Basic steps of AFLP fingerprinting

Double-stranded adapters consist of a core sequence and an enzyme-specific

Fig. 3. AFLP: Principle

Fig. 4. DNA sequencer

Fig. 5. AFLP electropherogram.

2. Random Amplified polymorphism DNA (RAPD)

Fig. 6. Pictorial view methodology of RAPD.

RAPD polymorphisms can theoretically result from several types of events:

Fig. 8. A Comparison of Plant DNA Typing Methods

Can RAPD Analysis Help Catch a Killer?

Fig. 9. Palo verde tree (Cercidium spp.)

Types of samples and collection for DNA

IV. Seeds, fruits and roots

Experimental methodologies in NHFG

Random Amplified Polymorphic DNA (RAPD) in Molecular Ecology.”

Genetic Relationships in the Tribe Datureae (Solanaceae).” Theoretical and

Milligram Amounts of Fresh, Herbarium and Mummified Plant Tissues.”

Science International 172 (2–3): 161–63.

You might also like