Download Genetic (molecular) Markers and their uses

Genetic (molecular) Markers
and their uses
What are molecular markers?
An identifiable physical location on a chromosome (e.g.,
restriction enzyme cutting site, microsatellite, random amplified
fragment polymorphism) or an isozyme, presenting
polymorphism among individuals, populations or taxa and
whose inheritance can be monitored.
A genetic marker is a gene or DNA sequence or a nucleotide,
with a known location on a chromosome, heritable as a simple
Mendelian trait, that can be used to identify individuals or
species.
Markers can exhibit two modes of inheritance:
a) dominant/recessive
or
b) co-dominant.
If the genetic pattern of homozygotes can be distinguished from
that of heterozygotes, then a marker is said to be co-dominant.
Generally co-dominant markers are more informative than the
dominant markers.
Ausefulmolecularmarkermustpossesscertaincharacteris7cs:
• Polymorphic: A polymorphism is a detectable and heritable
varia6onatalocus.
• Amarkerispolymorphicifthemostabundantallelecomprisesless
thanX%ofallthealleles,usually95%.
• Reproducible:Shouldgivesimilarresultsindifferentexperiments
irrespec6veofthe6meandtheplace.
• Preferably displays co-dominant inheritance (both forms are
detectableinheterozygotes).
• Thedetec6onofthemarkermustbefastandinexpensive.
• Demonstratesmeasurabledifference(s)inexpressionbetweentrait
types and/or alleles of interest, early in the development of the
organism.
• Hasnoeffectonthetraitofinterestthatvariesdependingonthe
alleleatthemarkerloci.
Polymorphism
The use of Molecular Markers is based on naturally occurring
polymorphism.
An enormous amount of polymorphism is present in natural
populations. The fist time this was realised was in 1966 when it was
found that at least 30% of the assayed loci in wild Drosophila
pseudoobscura encoded electrophoretic polymorphic proteins.
Base pair changes are more frequent than large rearrangements and
heterogeneity is not restricted by coding regions.
Differences at the DNA level vary between different species:
1-2 base pairs per 1000 in humans
more than 40 per 1000 in maize
Level of polymorphism
The resolving power of genetic markers is determined by the
level of polymorphism detected, which is primarily affected by
electrophoresis
the mutation rate at the genomic sites involved. GelIndividual
1
Individual 2
Polymorphism
Variation at isozyme loci is caused by point mutations, which
occur at low frequency (<10-6 per meiosis).
Moreover, only mutations altering the net electric charge of
proteins can be detected, thereby even reducing the resolving
power of isozymes.
Level of polymorphism (2)
In contrast, mutations at minisatellite and microsatellite loci,
mainly due to changes in the number of repeat units of the core
sequence, have been estimated to occur at the relatively high
frequency of 10-3-10-2 and 10-5-10-2 per meiosis, respectively.
In choosing the appropriate technique, the level of
polymorphism generally detected by the marker needs to be
considered in relation to the presumed degree of genetic
relatedness within the material to be studied.
In general, higher resolving power is required when samples
are more closely related.
How does mutations accumulate in plants
Plants face unique obstacles to long-term genetic integrity. They lack
reserved germ lines: gametes arise from meristem cells that have
already divided many times.
During meristem growth and subsequent floral development, DNA
integrity is jeopardized by multiple opportunities for replication errors
and for DNA damage by environmental mutagens from which plants
cannot escape, such as solar UV-B light and genotoxic chemicals, and
from endogenous DNA-damaging oxyradicals arising from
photosynthesis by chloroplasts and oxygen metabolism in
mitochondria.
Seed production versus vegetative propagation
Monoicy versus dioicy
Do plants evolve differently?
- Yes
1
because they do not posses a germline
2  because many of them grow to very large size (i.e. have
time to accumulate somatic mutations)
3  because plants grow in modular fashion, opening the
possibility for within ‘individual’ selection + variation
4  hierarchical levels of selection needs more attention in
population genetics
Evidence for somatically derived variation
Agricultural and horticultural varieties
pink Fosters grapefruit
normal
DNA Markers
RFLPs
RFLP refers to the variation among individuals in the lengths of
DNA fragments produced by restriction enzymes that cut DNA
at specific sites. The variations are due to differences in the
DNA sequence at these sites . (co-dominants)
Screening of RFLP polymorphism
DNAs of three maize lines were digested by Hind III (lines 1 to 3) and Eco RI
(lines 4 to 6) (M : molecular weight marker).
A : simple pattern : the probe corresponds to a single copy sequence.
B : complex pattern : the probe reveals several loci.
C : high background : the probe corresponds to a repeated sequence.
A family pedigree made from RFLPs
The pedigree shows the inheritance of a RFLP marker through three generations in
a single family. A total of 8 alleles (numbered to the left of the blots) are present in
the family. The RFLPs of each member of the family are placed directly below his
(squares) or her (circles) symbol and RFLP numbers.
Microsatélites
São repetições, no DNA, de unidades de 1 a 6 pares de bases.
O número de repetições varia entre 8 e 50 (5 –100).
Ocorrem geralmente em zonas não codificantes do genoma.
Nos organismos eucariotas podem encontrar-se 104 a 105 loci
de microsatélites no genoma.
Os níveis de mutação dos microsatélites são muitos altos e
variam entre 1x10-5 e 1x10-7/locus/geração.
As mutações nos microsatélites resultam:
1 - do deslizamento da polimerase do DNA, durante a
sua replicação, seguido da sua reparação;
2 - de mutações pontuais.
A
B
A – Origem dos polimorfismos revelados pela amplificação de microsatélites
B – Esquema de um gel de electroforese
Electroforese em gel de poliacrilamida 8%
(ALFexpress):
300 bp
288 bp
250 bp
220 bp
200 bp
ReproGel High Resolution; Voltagem- 1500V; Corrente- 60 mA
Potência- 30 W; Temperatura- 55 ºC
Vital
Trincadeira Preta
Touriga Nacional
Tinto Cão
Síria
Sercial
Rufete
Rabo de Ovelha
Moscatel Setúbal
Malvasia Fina
Castelão
Baga
Arinto
Aragonez
350 bp
Fernão Pires
- Padrão externo (50-500 bp)
- Padrões internos (fragmentos amplificados do M13mp18)
Moscatel de Setubal
Aragonez
Arinto
Castelão
Tinto Cão
Sercial
Trincadeira Prêta
Rabo de Ovelha8
Síria
Baga
Malvasia Fina
Fernão Pires
Vital
Rufete
0.1
Touriga Nacional
Vantagens do uso de Microsatélites
como marcadores genéticos
Específicos para um determinado locus.
Co-dominantes (podem-se distinguir os indivíduos
homozigóticos dos heterozigóticos).
São neutros.
Revelam-se por amplificação por PCR.
São muito polimórficos: apresentam um elevado número de
alelos de baixa frequência, denominados “alelos raros”.
5 microsatélites, cada um com 5 alelos igualmente frequentes
podem, estatisticamente, produzir 700.000 genótipos
diferentes.
Desvantagens
Custos de descoberta e optimização de amplificação eram
elevados. Agora já não com as NGS
Os indivíduos heterozigóticos podem ser classificados
como homozigóticos quando ocorrem alelos nulos devido
a mutações nos locais de emparelhamento dos “primers”
Bandas subsidiárias (“Stutter bands”)podem complicar a
identificação precisa de polimorfismos.
Os modelos mutacionais subjacentes ainda não estão
devidamente comprovados (“infinite alleles model” or
“stepwise mutation model”).
Homoplasia devido a mutações aumentando ou
diminuindo o nº de duplicações pode induzir a
submestimação de divergências genéticas.
AFLP-PCR or just AFLP
A PCR-based tool used in gene6cs research, DNA fingerprin6ng,
andintheprac6ceofgene6cengineering.
Developed in the early 1990s by Keygene, AFLP uses restric6on
enzymestodigestgenomicDNA,followedbyliga6onofadaptors
to the s6cky ends of the restric6on fragments. A subset of the
restric6on fragments is then selected to be amplified. This
selec6on is achieved by using primers complementary to the
adaptor sequence, the restric6on site sequence and a few
nucleo6des inside the restric6on site fragments (as described in
detailbelow).
The amplified fragments are separated and visualized on
denaturingpolyacrylamidegels,eitherthroughautoradiographyor
fluorescence methodologies, or via automated capillary
sequencinginstruments.
AAFLPgelprofilefor94F6individualsfromacrossofL.japonicus
RAPD
Standsforrandomamplifica7onofpolymorphicDNA.
It is a type of PCR reac6on, but the segments of DNA that are
amplifiedarerandom.
The scien6st performing RAPD creates several arbitrary, short
primers (8–12 nucleo6des), then proceeds with the PCR using a
large template of genomic DNA, hoping that fragments will
amplify.Byresolvingtheresul6ngpa\erns,asemi-uniqueprofile
canbegleanedfromaRAPDreac6on.
No knowledge of the DNA sequence for the targeted gene is
required, as the primers will bind somewhere in the sequence,
butitisnotcertainexactlywhere.
RAPDs
M OPC-04
OPC-07
M OPC-08
OPC-09
M
?
From left to right: Galega, Cordovil de Serpa, Verdeal
Alentejana with OPC-04, OPC-07, OPC-08, OPC-09,
respectively. M: 100bp ladder. (Operon Primers serie C)
ISSR
Inter-SSR is a PCR-based multilocular detection of SSRbased genetic polymorphisms without the need to discover the
unique genomic sequences flanking each repeats
REMAP
(Retrotransposon-Microsatellite Amplified Polymorphism)
Dominant, multiplex marker system that examines
variation in retrotransposon insertion sites. REMAP
fragments between retrotransposons and microsatellites are
generated by PCR, using one primer based on a LTR target
sequence and one based on a simple sequence repeat motif.
Long Terminal Repeat
SNPs – Single Nucleotide Polimorphisms
SNPs and Haplotypes
SNPs identified in a 263-nt
segment of the maize stearoylACP desaturase gene.
32 individuals sequenced.
The vertical columns identify
nine polymorphic sites, including
one insertion/deletion (I/D)
polymorphism.
4 distinct haplotypes are shown.
development of molecular marker techniques and their
advancements over last two decades
SRAP – Seq. Related Amplified Polimorph
Estimation of genetic variation in plants using
molecular techniques
Pratical applications of Plant Molecular Biology (1997) R.J. Henry (Ed.) Chapman&Hall
The kinds of things we will want to know using
Molecular Markers range from:
Individualization: does the pork carcass in the freezer
individually match the evidence in the field?
to Relatedness: can kin selection (high relatedness) explain
cooperative courtship display in tropical birds?
to Assignment to population source: do maritime pine
populations across Portugal show sufficient differentiation
to allow us to assign unknown samples to a source area
(with reasonable to high confidence)?
to Population genetic structure: what forces could explain the
observed patterns of genetic differentiation among
populations of a species (bears, olive trees, species of your
choice)?
to Species boundaries: are these two forms of roses a single
species or two distinct species?
to Phylogenetic trees: where do cetaceans fit in a phylogenetic
tree of mammalian groups?
to what is the Grand arrangement of the Tree of Life in terms
of kingdoms and phyla.
Why do we want this sort of information? The
reasons will range from:
Legal/forensic (environmental crimes or other);
to Answering Evolutionary Puzzles (how or why does this
behavior or mating system occur?);
to Assessing plant, fisheries or wildlife populations for
management reasons;
to Solving conservation problems — which taxa should we
focus on for conservation, where do areas of high endemism
occur, what are the consequences of anthropogenic (humaninduced) changes?
Applications of Genetic Markers to plants
Development of sampling strategies
Identification of collection gaps
Identification/validation of redundant germplasm
Quantification of genetic drift/shift
Identification of genetic contamination
Genetic evaluation of germplasm
Assembly/validation of core collections
Analysis of functional diversity
Distribution of genetic variation
Development of management strategies
Maintenance of genetic resources for utilization
A conceptual flow chart for molecular marker
analysis
Gels - DNA
patterns
Diversity
Indexes
Binary
Matrix
Haplotype
definition
AMOVA
(Analysis of
Molecular
Variance)
Genetic
Diversity
Jaccard
similarity
coefficient
Pearson
Correlation
coefficient
Principal
Component
Analysis (PCA)
Similarity
matrix
UPGMA / Neighbor Joining
Dendograms
Phenetic Relationships
Population genetic
structure
Bionumerics; NTSYS, Arlequin
Example: the Galega cultivar
MW
1
2
3
4
5
6
7
8
9
RAPDs and ISSRs
ISSR – (GACA)4
Ribatejo-Santarém
Ribatejo-Abrantes
A8
A2
A16
A15
B4
B3
B10
B1
B5 B6
A13
B2
A7
A4A14
C8
A5
C4
A11A9 A1
A10A6
A12
A3
A18
A17
C10
C5
C9
C7
Baixo-Alentejo
D22 D23
D21
D24
B8
B7
77 genotypes
C6
C3
C2
C1
D20
D19
D2
D26 D13D25
D5D4 D3
D17
D15 D6
D1
D16
D11
D10
D14D18
D12
D8
B9
Beira Litoral
Phenetic relationship
D9
D7
Genetic structure
E13
E5
Y
X
Z
E8
E1
E6 E3
E10
E2
E7
E9 E11E4
E12
Alto-Alentejo
Diversity and possible
origin of the cultivar
J(58%); CC(88%); B(49%)
J(53%); CC(85%); B(59%)
J(62%); CC(83%); B(73%)
J(47%); CC(88%); B(96%)
J(55%); CC(85%); B(64%)
J(34%); CC(93%); B(96%)
J(57%); CC(88%); B(100%)
J(31%); CC(93%)
J(38%); CC(92%); B(77%)
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11
A12
A13
A14
A15
A16
A17
A18
B1
B2
B3
B4
B5
B6
B7
B8
B9
B10
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
D1
D2
D3
D4
D5
D6
D7
D8
D9
D10
D11
D12
D13
D14
D15
D16
D17
D18
D19
D20
D21
D22
D23
D24
D25
D26
E1
E2
E3
E4
E5
E6
E7
E8
E9
E10
E11
E12
E13
Region III
Region IV
Region II
Dendrogram of similarity
among 77 accessions of
Galega cultivar
RAPDs and ISSR markers
using Jaccard coefficient.
Agglomeration method was
UPGMA.
Values at nodes represents
in percentage:
1- Jaccard similarity (J),
Region V
2 - Cophenetic Correlation
(CC) and
3 - Bootstrap analysis (B)
respectively.
Region I
Avaliação da estrutura genética dos zambujeiros
Análise da Variância Molecular (AMOVA)
Entre as populações
(7%)
Dentro das
populações
(93%)
p = 0.07
Diferenciação das diferentes populações baseada na estatística φ
Cobrançosa
Alf.Fé
Ag.
Zambujeiros
Alc.
Foz Côa Mac.Cav. Mirand.
0.287*
0.198*
0.190*
0.292*
Zambujeiros
Mog.
Valp.
Ag.
0.307*
0.101
0.000
Alc.
S.Olaia Mag.
0.535*** 0.462*** 0.451*** 0.545*** 0.549*** 0.413*** 0.127** 0.000
S.Olaia 0.617*** 0.519**
0.472** 0.586*** 0.659*
0.420*
0.063 0.162* 0.000
0.579** 0.478**
0.436**
0.556**
0.618*
0.386*
0.000
0.088
0.000
Ferest. 0.576** 0.479**
0.423**
0.542**
0.627*
0.380*
0.000 0.087* 0.094
0.000
Mag.
Ferest. S.Arr.
S.Arr. 0.427*** 0.354*** 0.375*** 0.455*** 0.431*** 0.311*** 0.047
0.020
0.000 0.100* 0.000
0.000
0.000 0.000
Nível de significância estatística: *** p = 0.001; ** 0.001< p < 0.01; * 0.01 < p < 0.05
As populações das duas variedades botânicas são distintas entre si
Ligeira diferenciação entre algumas das populações de zambujeiro
Não se detectou diferenciação entre as populações de ‘Cobrançosa’
Castascul7vadasevinhaselvagem:relaçõesgené7cas
0.35
0505
0203
0.14
Samar
Terra
UvaSal
Verdelh
Barc
Sauvi
TrebTo
_0116
_0210
_0204
_0117
_0109
_0208
_0214
_0122
_0114 _0502
_0509
_0120
_0206
_0112 _0213
_0209
_0201
_0121
_0108 _0101
_0111
_0212
_0102
_0410
_0118
_0110
_0119_0103
_0104 _0115
Syrah Encru
_0106
EspMol
_0512
_0105
Alvalh
_0125
UCao
Gouv
Monv
RieslB
TCaiDDam
Bical Mul-ThuB
CorGa
APreto
Molar TNacTPratas
_0107
Alvadu Aves
Dour
Cornife
Arag
Rufe
TGros
Cidre
Jaen Basta
Síria
UCav
Avaz
TDou
Teintur
Camar
AlicBr
FPire
Corr
CercB
More AriDou
NMolR
AlVer
Jamp
RabOvB
ArintoB
MFina
Laria
B
0411
Luzi
Verll
AramN
2 -0.08
A
0404
0412
0506
0407
0401
0406
0511 0403 0405
0507 04020503
D
Folg
0207
0408
0409
Ferral
CaberF
MRei
CoarNea
TFran
Semil
TriN
C
MantB
Maruf
TamarBastT
SNov AliBous Diag
BRati
Perr
Cast
MalCand
-0.29
MantT
-0.50
-0.60
-0.39
-0.17
1
0.04
0.25
Pinus pinaster
Population Analysis
Corsega–24
Cuenca-24
Ga82-24
Landes–25
PA18-24
PA54-24
Pb42-24
Pcse102–24
Pff06–24
PI20–8
PI24–8
PI26–8
Pm33–8
Pm34–8
Pm36–8
Pma29–12
Pma30–12
Pmb39–24
Pmc43–25
Pmg12–8
Pmg46-8
Pmg52–8
Pmt40–24
PS45–24
Psp02–24
exceptuando-se as
árvores da população
da Córsega (a
maioria encontrada
no grupo genético
“azul”, as restantes
árvores encontram-se
distribuídas
aleatoriamente pelos
diferentes grupos
genéticos.
Population Assignment
-15,000
-13,000
-11,000
-9,000
-7,000
Pop1
Med
Pop2
Atlant
Pop3
Escaroupim
-5,000
-4,000
-6,000
-8,000
Pop 2
-10,000
Pop1
Pop2
-12,000
-14,000
-16,000
Pop 1
-18,000
Pop3