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MOLECULAR MARKER TECHNOLOGIES

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Title: MOLECULAR MARKER TECHNOLOGIES


1
MOLECULAR MARKER TECHNOLOGIES
  • Training Workshop on Forest Biodiversity
  • 5-16 June 2006

Lee Soon Leong Forest Research Institute Malaysia
2
Outlines
  • Organization and flow of genetic information
  • Molecular techniques to reveal genetic variation
  • Type of molecular markers
  • Which marker for what purpose
  • Microsatellite marker
  • Case study 1 using microsatellites to estimate
    gene flow via pollen
  • Case study 2 using microsatellites for
    individual-specific DNA fingerprints

3
FLOW OF GENETIC INFORMATION
4
Deoxyribonucleic Acid (DNA) The molecule that
encodes genetic information
A pairs with T C pairs with G
DNA molecule consists of two strands that wrap
around each other to resemble a twisted ladder
5
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6
  • Organisms genomic DNAs are subjected to mutation
    as a result of normal cellular operations or
    interactions with environment

7
  • Mutations in genomic DNA can be classified into
    several categories

8
Through long evolutionary accumulation, many
different instances of mutation as mentioned
above should exist in any given species
The number and degree of the various types of
mutations define the genetic diversity within a
species
It has been widely recognized that loss of
genetic diversity is a major threat for the
maintenance and adaptive potential of species
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10
  • For many plant species, ex situ and in situ
    conservation strategies have been developed to
    safeguard the extant of genetic diversity
  • To manage this genetic diversity effectively the
    ability to identify genetic diversity is
    indispensable
  • In addition, for this variation to be useful, it
    must be heritable and discernable as
    recognizable phenotypic variation or as genetic
    mutation distinguishable through molecular marker
    technologies

11
Definition of molecular markers
A sequence of DNA or protein that can be screened
to reveal key attributes of its state or
composition and thus used to reveal genetic
variation
12
  • Four major molecular techniques are commonly
    applied to reveal genetic variation. These are
  • Polymerase chain reaction (PCR)
  • Electrophoresis
  • Hybridization
  • DNA sequencing

13
POLYMERASE CHAIN REACTION
The method was invented by Kary Banks Mullis in
1983, for which he received the Nobel Prize in
Chemistry ten years later
three temperature-controlled steps
14
ELECTROPHORESIS
Technique for separating the components of a
mixture of charged molecules (proteins, DNAs, or
RNAs) in an electric field within a gel or other
support
Migration rate depend on electrical charge and
size
15
HYBRIDIZATION
One of the most commonly used nucleic acid
hybridization techniques is Southern blot
hybridization
Southern blotting was named after Edward M.
Southern who developed this procedure at
Edinburgh University in the 1975
16
SEQUENCING
In 1977, 24 years after the discovery of the
structure of DNA, two separate methods for
sequencing DNA were developed chain termination
method and chemical degradation method
17
Recent detection techniques
TaqMan a probe used to detect specific
sequences in PCR products by employing 5 to 3
exonuclease activity of the Taq DNA polymerase
Pyrosequencing refers to sequencing by
synthesis, a simple to use technique for accurate
analysis of DNA sequences
Microarray Technology a high throughput
screening technique based on the hybridization
between oligonucleotide probes (genomic DNA or
cDNA) and either DNA or mRNA
18
TYPES OF MOLECULAR MARKERS
  • Due to rapid developments in the field of
    molecular genetics, a variety of molecular
    markers has emerged during the last few decades

19
Allozyme (biochemical marker)
  • The alternative forms of a particular protein
    visualized on a gel as bands of different
    mobility. Polymorphism due to mutation an amino
    acid has been replaced, the net electric charge
    of the protein may have been altered

Technique Electrophoresis and enzyme staining
20
RFLP (Non-PCR based marker)
  • Targets variation in DNA restriction sites and in
    DNA restriction fragments. Sequence variation
    affecting the occurrence (absence or presence) of
    endonuclease recognition sites is considered to
    be main cause of length polymorphisms

Techniques Electrophoresis and hybridization
21
RAPD (PCR-based marker)
Uses primers of random sequence to amplify DNA
fragments by PCR. Polymorphisms are considered to
be primarily due to variation in the primer
annealing sites, but they can also be generated
by length differences in the amplified sequence
between primer annealing sites
Techniques PCR and Electrophoresis
22
AFLP (PCR-based marker)
  • A variant of RAPD. Following restriction enzyme
    digestion of DNA, a subset of DNA fragments is
    selected for PCR amplification and visualization

Techniques PCR and Electrophoresis
23
Microsatellite (PCR based marker)
  • Targets tandem repeats of a small (1-6 base
    pairs) nucleotide repeat motif. Polymorphism due
    to the number of tandem repeats

Techniques PCR and Electrophoresis
24
  • Other markers
  • Cleaved Amplified Polymorphic Sequence
    (CAPS/PCR-RFLP)
  • Inter Simple Sequence Repeat (ISSR)
  • Single-strand conformation Polymorphism (SSCP)
  • Sequence Characterized Amplified Region (SCAR)
  • More recent markers
  • Single-Nucleotide Polymorphism (SNP)
  • Retrotransposon-based markers
  • Sequence-Specific Amplified Polymorphism (S-SAP)
  • Inter-retrotransposon Amplified Polymorphism
    (IRAP)
  • Retrotransposon-Microsatellite Amplified
    Polymorphism (REMAP)
  • Retrotransposon-Based Insertional Polymorphism
    (RBIP)

25
Weising, K., Nybom, H., Wolff, K. and Kahl, G.
2005. DNA Fingerprinting in Plants, Priciples,
Methos, and Applications. 2nd Edition. CRC
Press, Boca Raton, Florida, USA.
Spooner, D., van Treuren, R. and de Vicente, M.C.
2005. Molecular markers for genebank
management. IPGRI Technical Bulletin No. 10.
International Plant Genetic Resources Institute,
Rome, Italy.
Henry, R.J. 2001. Plant Genotyping The DNA
Fingerprinting of Plants. CAB International
Publishing, Wallingford, U.K.
26
Markers differ with respect to important features
  • Genomic abundance
  • Polymorphism level
  • Locus specificity
  • Reproducibility
  • Technical requirements
  • Financial investment

27
  • Codominance or dominace

Dominant marker A marker shows dominant
inheritance with homozygous dominant individuals
indistinguishable from heterozygous individuals
Codominant marker A marker in which both alleles
are expressed, thus heterozygous individuals can
be distinguished from either homozygous state
28
None of the available techniques is superior to
all others for a wide range of applications, but
the key-question rather is which marker to use in
which situation
  • Within and among population variation Allozyme,
    SSR, AFLP and RAPD
  • Mating system study Allozyme or microsatellite
  • Estimating gene flow via pollen and seed
    Microsatellite (SSR)
  • Phylogeography cpSSR
  • Clonal identification AFLP or RAPD
  • Polyploidy multilocus dominant marker (AFLP)
  • Genetic Linkage Mapping AFLP, RAPD, Allozyme,
    RFLP, SSR, CAPS, SNP
  • Phlogenetic study conserve within species (DNA
    sequencing)

.
29
  • A framework for selecting appropriate techniques
    for plant genetic resources conservation can be
    referred to

Karp, A., Kresovich, B., Bhat, K.V., Ayad, W.G.
and Hodgkin, T. 1997. Molecular Tools in Plant
Genetic Resources Conservation A Guide to the
Technologies. IPGRI Technical Bulletin No. 2.
International Plant Genetic Resources Institute,
Rome, Italy
30
Microsatellite marker
  • What are microsatellite?
  • Where are microsatellites found?
  • How do microsatellites mutate?
  • Abundance in genome
  • Why do microsatellite exist?
  • Models of mutation
  • Development of microsatellite primers
  • Genotyping procedure
  • Advantages
  • Disadvantages
  • Applications

31
What are microsatellite?
  • Tandem repeated sequences with a 1-6 repeat motif
  • Dinucleotide (CT)6 - CTCTCTCTCTCT
  • Trinucleotide (CTG)4 - CTGCTGCTGCTG
  • Tetranucleotide (ACTC)4 - ACTCACTCACTCACTC
  • Synonymous to SSR and STR Depending on nature of
    repeat tract, SSR can further divided into four
    categories

Perfect repeat when repeat tract pure for one motif CTCTCTCTCTCT
Compound SSR when repeat tract pure for two motifs CTCTCTCACACA
Imperfect SSR if single base substitution CTCTCTACTCTCT
Region of cryptic simplicity if complex but repetitive structure GTGTCACAGAGT
32
Where are microsatellites found?
Majority are in non-coding region
33
How do microsatellites mutate?
  • Microsatellites alleles change rather quickly
    over time
  • E. coli 10-2 events per locus per replication
  • Drosophila 6 X 10-6 events per locus per
    generation
  • Human 10-3 events per locus per generation

34
Abundance in genome
35
Why do microsatellite exist?
  • Majority are found in non-coding regions thought
    no selective pressure as "junk" DNA?
  • Regulate gene expression and protein function,
    e.g., human diseases caused by expansions of
    polymorphic trinucleotide repeats in genes
    fragile X and myotonic dystrophy
  • In plant, high density of SSRs were found in
    close proximity to coding regions regulatory
    properties
  • High level of polymorphism a necessary source of
    genetic variation

36
Models of Mutation
  • Several statistics based on estimates of allele
    frequencies (e.g., Fst Rst) rely explicitly on
    a mutation model
  • Size matters when doing statistical tests of
    population substructuring

37
Development of microsatellite primers
  • Can be time consuming and expensive. May be
    obtained by screening sequence in databases or
    screening libraries of clones
  • Standard method to isolate microsatellites from
    clones
  • Creation of a small insert genomic library
  • Library screening by hybridization
  • DNA sequencing of positive clones
  • Primer design and PCR analysis
  • Identification of polymorphisms
  • This approach can be extremely tedious and
    inefficient for species with low microsatellite
    frequencies

38
  • Alternative strategies to overcome
  • Selective hybridization using nylon membrane
  • Selective hybridization using steptavidin coated
    beads
  • RAPD based
  • Primer extension

39
Genotyping procedure
PCR
40
  • The use of fluorescently labeled primers, combine
    with automated electrophoresis system greatly
    simplified the analysis of microsatellite allele
    sizes

41
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Advantages
  • Low quantities of template DNA required (10-100
    ng)
  • High genomic abundance
  • Random distribution throughout the genome
  • High level of polymorphism
  • Band profiles can be interpreted in terms of loci
    and alleles
  • Codominance of alleles
  • Allele sizes can be determined with an accuracy
    of 1 bp, allowing accurate comparison across
    different gels
  • High reproducibility
  • Different SSRs may be multiplexed in PCR or on
    gel
  • Wide range of applications
  • Amenable to automation

44
Disadvantages
  • High development costs in case primers are not
    yet available. Primers might be species specific
  • Heterozygotes may be misclassified as homozygotes
    when null-alleles occur due to mutation in the
    primer annealing sites
  • Stutter bands on gels may complicate accurate
    scoring of polymorphisms
  • Underlying mutation model (infinite alleles model
    or stepwise mutation model) largely unknown
  • Homoplasy due to different forward and backward
    mutations may underestimate genetic divergence

45
Applications
Generally, high mutation rate makes them
informative and suitable for intraspecific
studies but unsuitable for studies involving
higher taxonomic levels
  • Population genetics investigations within a
    genus of centers of origin, genetic diversity,
    population structures and relationships among
    species
  • Parentage analysis seed orchard monitoring,
    mating systems and gene flow via pollen seed
  • Fingerprinting clone confirmation and
    individual-specific fingerprints
  • Genome mapping - Constructing full coverage or
    QTL maps
  • Comparative mapping - Genome structure, framework
    maps, or transferring trait and marker data among
    species

46
Case study 1 Using microsatellites to estimate
gene flow via pollen
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48
Shorea parvifolia
Shorea leprosula
49
Methodology
50
Microsatellite Loci
No. of clones sequenced No. of clones with SSR () No. of unique SSR clones () Core sequence (no. of clones repeat times)
624 592 (94.9) 315 (53.2) CT/GA (266 84.4 6-78) GT/CA (29 9.2 8-46) Others (20 6.4 6-40)
Locus Primer sequence (5 3) Repeat motif Length N Size range He PIC
lep074a F ATC ACC AAG TAC CTA TCA TCA R GCA ATG GCA CAC AGT CTA TC (CT)11 124 11 110-130 0.824 0.791
lep079 F GTT GTC TGT TCT TAC CAG GAA G R GCA TAA GTA TCG TCG CCA (CT)11 162 13 155-198 0.830 0.798
lep111a F GGA AAC TAC TGG AGC AGA GAC R GGT GGG TTA TGG AGA ATG AG (GA)14 152 12 138-154 0.855 0.821
lep118 F AAA GCG TAC AAA TTC ATC A R CTA TTG GTT GGG TCA GAA GG (GA)16 170 15 145-176 0.892 0.861
lep280 F GCA ACT AAA ATG GAC CAG A R GAG TAA GGT GGC AGA TAT AGA G (CT)7 119 11 107-137 0.851 0.816
lep384 F CCA AGA CAA CTC AAT CCT CA R AGA TGA AGG TGT TGC TGT G (CT)13 206 14 191-219 0.657 0.632
lep562 F TGA TTT GGG TGG TTG TAG R TAT TAC ATT TTT CAA GTC AAG TC (GT)8 164 12 154-180 0.883 0.852
Lee, S.L. et al. 2004. Isolation and
characterization of 21 microsatellite loci in an
important tropical tree Shorea leprosula and
their applicability to S. parvifolia. Molecular
Ecology Notes 4 222-225
51
50 ha demographic plot in Pasoh Forest Reserve
52
Pasoh Forest Reserve - 50-ha plot (190
individuals of S. leprosula and 102 of S.
parvifolia ? 27 cm dbh within the 50-ha plot)
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  • Shorea leprosula 9 loci (Pe 0.999)
  • lep074a, lep384, lep111a, lep118, lep280, lep267,
    lep294, lep475 lep562
  • PCR (500 x 9 4500 reactions)
  • Shorea parvifoila 6 loci (Pe 0.999)
  • lep074a, lep384, lep111a, lep118, lep280 lep294
  • PCR (360 x 6 2160 reactions)

57
S. leprosula (SL48)
MT48
58
S. parvifolia (SP35)
MT35
59
Mother tree (no. of seed analyzed) Mean distance between MT outcrossing (no. of seed) pollen outside plot Mean pollen flow distance
Shorea leprosula Shorea leprosula Shorea leprosula Shorea leprosula Shorea leprosula
SL048 (45) 267.1 ? 136.2 93.3 (42) 20.0 (9) 152.9 ? 99.6
SL062 (44) 363.2 ? 151.6 88.6 (39) 20.5 (9) 302.6 ? 188.9
SL074 (48) 259.2 ? 151.2 85.4 (41) 18.8 (9) 148.6 ? 187.2
SL075 (43) 292.6 ? 145.8 67.4 (29) 18.6 (8) 173.1 ? 103.8
SL084 (46) 512.6 ? 228.3 82.6 (38) 23.9 (11) 448.2 ? 245.3
SL109 (45) 343.7 ? 158.8 95.6 (43) 33.3 (15) 285.0 ? 154.5
SL160 (44) 567.1 ? 243.1 81.8 (36) 31.8 (14) 580.3 ? 288.4
Mean 372.2 ? 121.6 85.0 ? 9.3 23.8 ? 6.2 298.7 ? 164.0
Shorea parvifolia Shorea parvifolia Shorea parvifolia Shorea parvifolia Shorea parvifolia
SP009 (32) 309.0 ? 166.5 59.4 (19) 9.4 (3) 61.9 ? 100.5
SP014 (48) 307.7 ? 165.1 62.5 (30) 14.6 (7) 105.1 ? 140.9
SP020 (42) 348.7 ? 172.2 85.6 (36) 33.3 (14) 194.0 ? 146.7
SP022 (47) 239.6 ? 133.2 72.3 (34) 21.3 (10) 148.2 ? 125.0
SP025 (46) 376.2 ? 192.4 56.5 (26) 19.6 (9) 317.1 ? 277.0
SP035 (44) 244.2 ? 139.9 22.7 (10) 2.3 (1) 185.0 ? 159.7
Mean 304.2 ? 54.7 59.8 ? 21.1 16.8 ? 10.7 168.6 ? 88.1
60
Mother tree (no. of seed analyzed) Breeding unit parameters Breeding unit parameters Breeding unit parameters
Mother tree (no. of seed analyzed) Size (individual) Area (ha) Radius (m)
Shorea leprosula Shorea leprosula Shorea leprosula Shorea leprosula
SL048 (45) 203.6 63.6 450.1
SL062 (44) 208.0 65.0 454.9
SL074 (48) 205.0 64.1 451.6
SL075 (43) 221.0 69.0 468.8
SL084 (46) 225.2 70.4 473.3
SL109 (45) 245.7 76.8 494.4
SL160 (44) 261.8 81.8 510.3
Mean 224.3 ? 22.1 70.1 ? 6.9 471.9 ? 23.0
Shorea parvifolia Shorea parvifolia Shorea parvifolia Shorea parvifolia
SP009 (32) 81.9 59.4 434.7
SP014 (48) 90.0 65.2 455.6
SP020 (42) 112.9 81.8 510.3
SP022 (47) 97.8 70.8 474.8
SP025 (46) 105.5 76.5 493.4
SP035 (44) 76.7 55.6 420.5
Mean 94.1 ? 13.9 68.2 ? 10.1 464.9 ? 34.5
61
Negative exponential curve
y ae(-x/c)
62
Conclusion
  • Moderate pollen flow (150 300 m) Thrips as
    pollinators
  • Predominant outcrossing (85) mix-mating (60)
  • Model for pollen dispersal negative exponential
    model
  • Optimum population size for conservation -
    breeding unit area breeding unit size obtained
    (about 70 ha)

63
Case study 2 Using microsatellites for
individual-specific DNA fingerprints
64
In forensic applications in forestry and chain of
custody certification, two types of databases are
required
65
DNA markers to match the illegal log into its
original stump
66
  • However, In DNA testimony, it is necessary to
    provide an estimate of the weight of the evidence
  • Three possible outcomes of a DNA test no match,
    inconclusive, or MATCH between samples examined
  • If MATCH, it would not be scientifically
    justifiable to speak of a match as poor proof of
    identity in the absence of underlying data that
    permit some reasonable estimate of how rare the
    matching characteristics actually are
  • Therefore, in forensic casework, a population
    database must be established for statistical
    evaluation of the evidence to extrapolate the
    possibility of a random match

67
Neobalanocarpus heimii
68
Methodology
69
Sample collection
70
SSRs screening
  • 51 SSR primer pairs developed for dipterocarps
  • Neobalanocarpus heimii (6) (Iwata et al. 2000)
  • Shorea lumutensis (2) (Lee et al. 2006)
  • Shorea leprosula (21) (Lee et al. 2004a)
  • Hopea bilitonensis (15) (Lee et al. 2004b)
  • Shorea curtisii (7) (Ujino et al. 1998)

71
Specific amplification
72
Mode of inheritance
Qualitative observations (each progeny possessed
at least one maternal allele) to support the
postulation of single-locus mode of inheritance
73
Null allele
  • Homozygote excess (MICROCHECKER Van Oosterhout
    et al. 2004)
  • Examine patterns of inheritance
  • If any Individuals repeatedly fail to amplify any
    alleles at just one locus while other loci
    amplify normally

74
Repeat motif
Dinucleotide repeats (CT)n to mononucleotide
repeats (A)n
75
Size homoplasy
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  • What model to use product rule or subpopulation
    models?

Pasoh Forest Reserve (231 individuals)
  • Perform statistical tests to check
  • Hardy-Weinberg equilibrium for allele
    independence
  • Linkage equilibrium for locus independence
  • Results clearly showed that population is
    deviated from HWE

Population substructuring
Inbreeding
78
  • Random match probability need to be calculated
    using subpopulation model and corrected for
    coancestry (FST) and inbreeding (FIS)
    coefficients

79
Population structure of N. heimii throughout P.
Malaysia
80
DNA fingerprinting databases of N. heimiii
throughout P. Malaysia
Hardy-Weinberg equilibrium for allele
independence Linkage equilibrium for locus
independence
81
Applications of the databases
82
Genotypes Genotypes
Nhe004 262/262 262/262
Nhe005 129/129 129/129
Nhe011 176/186 176/186
Nhe015 143/181 143/181
Nhe018 141/169 141/169
Nhe019 214/220 214/220
Hbi016 140/141 140/141
Hbi161 102/105 102/105
Sle111a 137/140 137/140
Sle392 187/189 187/189
Sle605 120/120 120/120
Slu044a 148/148 148/148
Shc03 131/139 131/139
Shc04 85/117 85/117
Shc07 169/169 169/169
Shc09 190/201 190/201
Locus
83
Using database to extrapolate the possibility of
a random match
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