Download Parent 1

Marcatori genetici nel plant
breeding
Utilizzo
Identificazione clonale
Analisi dei parentali
Struttura familiare
Struttura di popolazione
Flusso genico
Filogeografia
Ibridizzazione
Filogenesi
Marcatori in biologia
Marcatori fenotipici = caratteri
distinguibili a occhio nudo
Colore del fiore, forma del baccello, etc..
P = E+G
Karl Von Linne (1707-1778)
Molecular markers
Sequenze di proteine o DNA facilmente distinguibili,
la cui eredità può essere verificata e associata con
tratti ereditati indipendentemente dall’ambiente :
a) Polimorfismi di proteine
b) Polimorfismi di DNA
Molecular markers
Sequenze (SNPs)
Microsatelliti (SSRs)
Multi-locus fingerprints
AFLP
(Amplified Fragment Length Polymorphism)
RAPD
(random amplified polymorphic DNA)
DNA cloroplastico PCR-RFLP
allozimi (elettroforesi di proteine)
Polimorfismi di proteine
Proteine di riserva del
seme
Isozimi
Isozimi
Isozimi
Starch gel of the isozyme malate dehydrogenase (MDH). The numbers
indicate first the MDH locus, and next the allele present (ie. 3-18 is locus
3 allele 18). Some bands are heterodimers (intralocus or interlocus).
Struttura del DNA
dal cromosoma al DNA
Stretch of nitrogen fixation gene in soybean
1 ccacgcgtcc gtgaggactt gcaagcgccg cggatggtgg gctctgtggc tgggaacatg 61 ctgctgcgag ccgcttggag
gcgggcgtcg ttggcggcta cctccttggc cctgggaagg 121 tcctcggtgc ccacccgggg actgcgcctg cgcgtgtaga
tcatggcccc cattcgcctg 181 ttcactcaga ggcagaggca gtgctgcgac ctctctacat ggacgtacag gccaccactc
241 ctctggatcc cagagtgctt gatgccatgc tcccatacct tgtcaactac tatgggaacc 301 ctcattctcg gactcatgca
tatggctggg agagcgaggc agccatggaa cgtgctcgcc 361 agcaagtagc atctctgatt ggagctgatc ctcgggagat
cattttcact agtggagcta 421 ctgagtccaa caacatagca attaaggtag gaggagggat ggggatgttg tgtggccgac
481 agttgtgagg ggttgtggga agatggaagc cagaagcaaa aaagagggaa cctgacacta 541 tttctggctt
cttgggttta gcgattagtg cccctctctc atttgaactc aactacccat 601 gtctccctag ttctttctct gcctttaaaa
aaaaatgtgt ggaggacagc tttgtggagt 661 ctgaaatcac catctacctt tacttaggtt ctgagtgcca aacccaaggc
accaggcatg 721 cgtccttgac tccggagcca tcaggcaggc tttcctcagc cttttgcagc caagtctttt 781 agcctattgg
tctgagttca gtgtggcagt tggttaggaa agaaggtggt tcttcgacca 841 ctaacagttt ggatttttta ggatgctagt
cctttaaaa ……….
molecular marker?
1 ccacgcgtcc gtgaggactt gcaagcgccg cggatggtgg gctctgtggc tgggaacatg 61 ctgctgcgag ccgcttggag gcgggcgtcg
ttggcggcta cctccttggc cctgggaagg 121 tcctcggtgc ccacccgggg actgcgcctg cgcgtgtaga tcatggcccc cattcgcctg 181
ttcactcaga ggcagaggca gtgctgcgac ctctctacat ggacgtacag gccaccactc 241 ctctggatcc cagagtgctt gatgccatgc
tcccatacct tgtcaactac tatgggaacc 301 ctcattctcg gactcatgca tatggctggg agagcgaggc agccatggaa cgtgctcgcc 361
agcaagtagc atctctgatt ggagctgatc ctcgggagat cattttcact agtggagcta 421 ctgagtccaa caacatagca attaaggtag
gaggagggat ggggatgttg tgtggccgac 481 agttgtgagg ggttgtggga agatggaagc cagaagcaaa aaagagggaa cctgacacta
541 tttctggctt cttgggttta gcgattagtg cccctctctc atttgaactc aactacccat 601 gtctccctag ttctttctct gcctttaaaa aaaaatgtgt
ggaggacagc tttgtggag
DNA
M1
Gene A
M2
MFG
Gene B
MFG
AACCTGAAAAGTTACCCTTTAAAGGCTTAAGGAAAAAGGGTTTAACCAAGGAATTCCATCGGGAATTCCG
readily detectable sequence of DNA whose inheritance can be
monitored and associated with the trait inheritance
Image from UV light
table
Image from computer
screen
Co-dominant marker
Gel configuration
P1
P2
O1
O2
Dominant marker
P2
O1
Polymorphism
Parent 1 : one band
Gel configuration
P1
Polymorphism
-Parent 1 : one band
-Parent 2 : a smaller band
-Offspring 1 : heterozygote =
both bands
-Offspring 2 : homozygote
parent 1
O2
-Parent 2 : no band
-Offspring 1 : homozygote parent 1
-Offspring 2 : ????
Dominant versus Co-dominant
Dominant:
No distinction between homo- and heterozygotes
possible
No allele frequencies available
AFLP, RAPD
Co-dominant:
homozygotes can be distinguished from
heterozygotes; allele frequencies can be calculated
microsatellites, SNP, RFLPs
Desirable properties for a good
molecular marker
* Polymorphic
* Co-dominant inheritance
* Occurs throughout the genome
* Reproducible
* Easy, fast and cheap to detect
* Selectivity neutral
* High resolution with large number of
samples
Basis for DNA marker technology
•Restriction Endonucleases
•Polymerase chain reaction (PCR)
•DNA-DNA hybridization
•DNA sequencing
RFLP based markers
*Examine differences in size of specific DNA
restriction fragments
*Require pure, high molecular weight DNA
*Usually performed on total cellular genome
Endonucleases and restriction sequences
Name
of the
enzyme
Number
of
cutting
sites
Taq I
MboI
Alu I
Dde I
Rsa I
Scrf I
Msp I
Hae
III
Ssp I
639
623
341
309
286
239
214
196
137
AG CT
C TNAG
GT AC
CC NGG
CC GG
GG
CC
AAT ATT
Cutting
sites
TCGA
GATC
Note: N represent any base : A, T, C or G
AAATCGGGACCTAATGGGCC
Ind 1
YFG
ATTTAGGGCAATTCCAAGGA
Ind 2
RFLP techniques
RFLP Polymorphisms interpretation
MFG
1
2
3
4
5
6
1
2
3
4
5
6
Advantages and disadvantages of RFLP
• Advantages
– Reproducible
– Co-dominant
– Simple
• Disadvantages
– Time consuming
– Expensive
– Use of radioactive
probes
DNA/DNA Hybridization
Denaturation
Elevated temperature
Known DNA sequence
Polymerase Chain Reaction
•Powerful technique for amplifying DNA
• Amplified DNA are then
separated by gel
electrophoresis
PCR based
methods
1. Reactions conditions
*Target DNA ( or template)
*Reaction buffer containing the co-factor MgCl2
*One or more primers
*Four nucleotides (dATP, dCTP, dGTP, dTTP)
*Thermostable DNA polymerase
2. Use of DNA polymerase
= an enzyme that can synthesize DNA at
elevated temperature
ex : Taq = enzyme purified from hot
spring bacterium : Thermus aquaticus
3. Thermal cycle
*Denaturing step - one to several min at 94-96 º C
*Annealing step - one to several min at 50-65 º C
*Elongation step - one to several min at 72 º C
4. Repetition
–typically 20 to 50 times average 35 times
AFLP Markers




Most complex of marker technologies
Involves cleavage of DNA with two
different enzymes
Involves ligation of specific linker pairs
to the digested DNA
Subsets of the DNA are then amplified
by PCR
AFLP Markers




The PCR products are then separated on
acrylamide gel
128 linker combinations are readily
available
Therefore 128 subsets can be amplified
Patented technology
AFLP Markers





Technically demanding
Reliable and stable
Moderate cost
Need to use different kits adapted to
the size of the genome being analyzed.
Like RAPD markers need to be
converted to quick and easy PCR based
marker
RAPD Markers




There are other problems with RAPD
markers associated with reliability
Because small changes in any variable
can change the result, they are unstable
as markers
RAPD markers need to be converted to
stable PCR markers.
How?
RAPD Markers




The polymorphic RAPD marker band is
isolated from the gel
It is used a template and re-PCRed
The new PCR product is cloned and
sequenced
Once the sequence is determined, new
longer and specific primers can be
designed
RAPD
•
Amplifies anonymous
stretches of DNA using
arbitrary primers
• Fast and easy method for
detecting polymorphisms
• Domimant markers
• Reproducibility
problems
RAPD Polymorphisms among landraces of sorghum
Sequences of 10-mer
RAPD primers
RAPD gel configuration
Name
Sequence
OP
OP
M
OP
OP
OP
5’
5’
5’
5’
5’
A08
A15
A 17
A19
D02
–GTGACGTAGG- 3’
–TTCCGAACCC- 3’
–GACCGCTTGT- 3’
–CAAACGTCGG- 3’
–GGACCCAACC- 3’
SSR repeats and primers
Repeat
GGT(5)
Sequence
GCGCCGAGTTCTAGGGTTTCGGAATTTGAACCGTC
GAGGGCTGATGAGGTGGATA
ATTGGGCGTCGGTGAAGAAGTCGCTTCCGTCGTTTGAT
TCCGGTCGTCAGAATCAGAATCAGAATCGATATGGTG
GCAGTGGTGGTGGTGGTGGTGGTTTTGGTGGTGGTGA
ATCTAAGGCGGATGGAGTGGATAATTGGGCGGTTGGT
AAGAAACCTCTTCCTGTTAG
ATCTTATGGCGGTTCTCGTG
ATTCTGGAATGGAACCAGATCGCTGGTCTAGAGGTTCT
GCTGTGGAACCA…..
SSR polymorphisms
P1
AATCCGGACTAGCTTCTTCTTCTTCTTCTTTAGCGAATTAGG
P2 AAGGTTATTTCTTCTTCTTCTTCTTCTTCTTCTTAGGCTAGGCG
P1
Gel configuration
P2
Linkage groups
SSR scoring for F 5:6 pop from the cross
Anand x N97-3708-13
M
4.
SNPs (Single Nucleotide Polymorphisms)
SNPs on a DNA strand
Hybridization using fluorescent dy
•Any two unrelated individuals differ by one base pair every
1,000 or so, referred to as SNPs.
•Many SNPs have no effect on cell function and therefore
can be used as molecular markers.
DNA sequencing
Sequencer
Sequencing gel
Sequencing graph
Types of traits =types of markers
Single gene trait: seed shape
MFG
Multigenic trait; ex: plant growth
=Quantitative Trait Loci
MFG
USES OF MOLECULAR MARKER

Measure genetic diversity
 Mapping
 Tagging
Genetic Diversity
Define appropriate geographical scales for
monitoring and management (epidemology)

Establish gene flow mechanism

identify the origin of individual (mutation
detection)
 Monitor the effect of management practices

manage small number of individual in ex situ
collection
 Establish of identity in cultivar and clones
(fingerprint)

paternity analysis and forensic

Genetic Diversity
Gotcha!
fingerprints
seeds,
plantlets
early selection
of the good allele
Mapping

The determination of the position
and relative distances of gene on
chromosome by means of their
linkage

Genetic map
A linear arrangement of genes or genetic markers
obtained based on recombination

Physical map
A linear order of genes or DNA fragments
Physical Mapping
It contains ordered overlapping
cloned DNA fragment
 The cloned DNA fragments are
usually obtained using restriction
enzyme digestion

QTL Mapping
A set of procedures for detecting
genes controlling quantitative traits
(QTL) and estimating their genetics
effects and location

 To assist selection
Marker Assisted Selection



Breeding for specific traits in plants
and animals is expensive and time
consuming
The progeny often need to reach
maturity before a determination of the
success of the cross can be made
The greater the complexity of the trait,
the more time and effort needed to
achieve a desirable result.
MAS



The goal to MAS is to reduce the time
needed to determine if the progeny
have trait
The second goal is to reduce costs
associated with screening for traits
If you can detect the distinguishing
trait at the DNA level you can identify
positive selection very early.
Developing a Marker



Best marker is DNA sequence
responsible for phenotype i.e. gene
If you know the gene responsible and
has been isolated, compare sequence of
wild-type and mutant DNA
Develop specific primers to gene that
will distinguish the two forms
Developing a Marker



If gene is unknown, screen contrasting
populations
Use populations rather than individuals
Need to “blend” genetic differences
between individual other than trait of
interest
Developing Markers




Cross individual differing in trait you
wish to develop a marker
Collect progeny and self or polycross
the progeny
Collect and select the F2 generation for
the trait you are interested in
Select 5 - 10 individuals in the F2
showing each trait
Developing Markers




Extract DNA from selected F2s
Pool equal amounts of DNA from each
individual into two samples - one for
each trait
Screen pooled or “bulked” DNA with
what method of marker method you
wish to use
Method is called “Bulked Segregant
Analysis”
Marker Development



Other methods to develop population
for markers exist but are more
expensive and slower to develop
Near Isogenic Lines, Recombinant
Inbreeds, Single Seed Decent
What is the advantage to markers in
breeding?
Reducing Costs via MAS

Example disease resistance
• 10000 plants

Greenhouse space or field plots
• $5000 - $10000

Time 4 months (salary)
• $10 - $15000
• total cost = $15 - $25,000
Reducing Costs via MAS






PCR-based testing @ $5 sample
$50,000 - costs more?
Analysis of trait not easily phenotyped
E.g: Cadmium in Durum wheat
10000 plants need to reach maturity
Cadmium accumulates in seed
Reducing costs via MAS






$15 - 25 growing costs + analysis
Atomic Absorption @ $15 per sample
$150,000 + growing costs
PCR analysis still $50000
Savings in time and money increase as
more traits are analyzed
Many biochemical tests cost >$50
sample
Marker Assisted Breeding



MAS allows for gene pyramiding incorporation of multiple genes for a
trait
Prevents development of biological
resistance to a gene
Reduces space requirements - dispose
of unwanted plants and animal early
QTL study
Trait M. 1 M. 2 M. 3
P.1
P.2
I.1
I.2
I.3
I.4
2.5
8.4
7.1
2.5
4.5
2.3
1
3
3
2
2
1
1
3
1
1
3
1
1
3
1
1
2
3
Statistical programs used in molecular marker studies
* SAS
* ANOVA
* Mapmaker
* Cartographer
Types of population used for molecular markers studies:
F2, RILs, Backcrosses (MILs), DH.
QTL Mapping