Diapositiva 1

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Marker Assisted Selection (MAS)
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DNA sources

1. Genomic DNA from chromosomes (Fragments because
   usually too large to clone directly)
2. cDNA (complementary DNA) derived by action of
   reverse transcriptase from (usually) mRNA
   template
3. chemically synthesized DNA molecules
   (oligonucleotides)

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Types of markers

• Morphological markers
• Seed color e.g. Kernel color in maize
• Function based e.g. Plant height associated with
  salt tolerance in rice

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• Limitations
• Most phenotypic markers are undesirable in the
  final product (Yellow color in maize).
• Dominance of the markers homozygotes/
  heterozygotes not distinguishable
• Sometimes dependent on the environment for
  expression e.g. Height of plants

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• Molecular markers
• Non-DNA such as isozyme markers Restricted due
  limited number of enzyme systems available.
• DNA based markers Markers based on the
  differences in the DNA profiles of individuals.

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• Some molecular markers are pieces of DNA that
  have no know function or impact on plant
  performance (Linked Markers)
• Detected via mapping.
• Linked markers are near the gene of interest and
  are not part of the DNA of the gene.
• Other markers may involve the gene of interest
  itself (Direct Markers)
• Based on part of the gene of interest.
• Hard to get but great once you have it.

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What is MAS?

• Concept of using molecular markers particularly
  DNA based to detect and track presence of gene
  transfer in breeding programs
• MAS works on the principle of linkage
  dis-equilibrium where markers that are tightly
  linked to target genes segregate together in a
  non random manner (due linkage)

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Advantages of MAS
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1. Improvement of response to selection (Rs)
2. Assays require small amount of tissue, therefore
   no destructive sampling.
3. Use of codominant markers allows accurate
   identification of individuals for scoring without
   ambiguity
4. Multiple sampling for various QTLs is possible
   from same DNA prep
5. Can assay for traits before they are expressed,
   e.g. before flowering
6. Time saving.

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CONVENTIONAL PLANT BREEDING
P2
P1
x
Donor
Recipient
F1
large populations consisting of thousands of
plants
F2
PHENOTYPIC SELECTION
Phosphorus deficiency plot
Salinity screening in phytotron
Bacterial blight screening
Field trials
Glasshouse trials
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MARKER-ASSISTED BREEDING
P2
P1
x
Resistant
Susceptible
F1
large populations consisting of thousands of
plants
F2
Method whereby phenotypic selection is based on
DNA markers
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Overview of marker genotyping
(1) LEAF TISSUE SAMPLING
(2) DNA EXTRACTION
(3) PCR
(4) GEL ELECTROPHORESIS
(5) MARKER ANALYSIS
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Requirements for a useful molecular marker

1. Molecular markers must be tightly linked to a
   target gene. The linkage must be really tight
   such that the presence of the marker will
   reliably predict the presence of the target gene.
2. The marker should be able to predict the presence
   of the target gene in most if not all genetic
   backgrounds.

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MAS
Marker 1 (more tightly linked than 2)
1

2
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Markers must be tightly-linked to target loci!

• Ideally markers should be lt5 cM from a gene or QTL

• Using a pair of flanking markers can greatly
  improve reliability but increases time and cost

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-
Plant A
Marker
Plant B
Plant C
Monomorphic bands
Polymorphic bands

Presens of a band, 1
Absence of a band, 0
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Markers must be polymorphic
RM84
RM296
1 2 3 4 5 6 7 8
1 2 3 4 5 6 7 8
P1 P2
P1 P2
Not polymorphic
Polymorphic!
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DNA extractions
Mortar and pestles
Porcelain grinding plates
LEAF SAMPLING
Wheat seedling tissue sampling in Southern
Queensland, Australia.
DNA EXTRACTIONS
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PCR-based DNA markers

• Generated by using Polymerase Chain Reaction
• Preferred markers due to technical simplicity and
  cost

PCR Buffer MgCl2 dNTPS Taq Primers DNA
template
PCR
THERMAL CYCLING
GEL ELECTROPHORESIS Agarose or Acrylamide gels
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Agarose gel electrophoresis
UV transilluminator
UV light
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A woman is uncertain which of two men is the
father of her child. DNA typing is carried out on
blood from the child (C), the mother (M), and
each of the two males (A and B), using probes for
a highly polymorphic DNA marker on two different
chromosomes (locus 1 and locus 2). The result
is shown in the accompanying diagram.
Can either male be excluded as the possible
father? Explain your reasoning.
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DNA replication in natural systems requires

1. A source of the nucleotides adenine (A), cytosine
   (C), thymine (T), and guanine (G).
2. The DNA polymerase (DNA synthesis enzyme).
3. A short RNA molecule (primer).
4. A DNA strand to be copied.
5. Proper reaction conditions (pH, temperature).

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• Is there any differences between PCR and
  mechanisms of the natural replication system?
• DNA primers are used instead of the RNA primer
  found in the natural system.
• Magnesium ions that play a role in DNA
  replication are added to the reaction mixture.
• A DNA polymerase enzyme that can withstand high
  temperatures, such as Taq, is used.
• A reaction buffer is used to establish the
  correct conditions for the DNA polymerase to work.

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Conditions under which MAS is valuable

1. Low heritability traits

• Traits too expensive to score Soybean Cyst
  Nematode (SCN) resistance. Young (1999)
• Recessive genes Pyramiding of dominant and
  recessive genes conferring resistance to
  important crop diseases which would otherwise be
  very difficult
• Multiple genes (Quantitative traits) QTLs
  underlying phenotypic and physiological traits
  can be traced using markers. Although QTL mapping
  is tedious, markers once identified can be used
  fast and accurately to detect the QTLs of
  interest.

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1. Quarantine No need to grow plants to screen for
   viral diseases that can not be visually detected,
   and small tissues can be used for DNA typing.

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Limitations of MAS

1. Cost of equipment, reagents and personnel.
2. Data collected in the field is assumed to be
   normally distributed, but usually is not.
3. Integration of the DNA information into existing
   systems is difficult.
4. Linkage drag. As the marker distance from the
   target gene increases, more of the donor DNA is
   retained in the desired background resulting in
   need for more backcrosses.

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Types of DNA based Markers
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• Hybridization based markers
• Restriction Fragment Length Polymorphisms (RFLPs)
  where differences in the number and size of
  fragments is analyzed

• Polymerase Chain Reaction (PCR) based
• Randomly Amplified Polymorphic DNAs (RAPDs),
  Single Sequence Repeats (SSRs).
• Amplified Fragment Length Polymorphic DNA (AFLPs)
  
• Other variants such as SCAR, CAPS, SSCP e.t.c.

• Sequence based markers
• Expressed Sequence Tags (ESTs),
• Single Nucleotide Polymorphism (SNPs)

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Restriction Fragment Length Polymorphisms (RFLPs)

• The first type of DNA markers that were used for
  genetic mapping were RFLPs.
• For instance a given restriction site may be
  present in one line and not in the other.

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• Procedure
• For detecting RFLPs involves the fragmentation of
  genomic DNA by a Restriction enzyme, which can
  recognize and cut DNA wherever a specific short
  sequence occurs.
• The resulting DNA fragments are then separated by
  length in agarose gel electrophoresis, and
  transferred to a membrane via the Southern blot
  procedure.
• Hybridization of the membrane to a labeled DNA
  probe then determines the size of the fragments
  which are complementary to the probe.

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• An RFLP occurs when the size of a detected
  fragment varies between individuals.
• Each fragment size is considered an allele, and
  can be used in genetic analysis.

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• RFLP markers have several advantages
• They are co-dominant and unaffected by the
  environment.
• Any source DNA can be used for the analysis.
• Many markers can be mapped in a population that
  is not stressed by the effects of phenotypic
  mutations.

• A main disadvantage is that
• RFLP mapping necessitates relatively large
  amounts of DNA.

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Cleaved Amplified Polymorphic Sequences (CAPS)

• The principle of CAPS markers is very similar to
  that of RFLP markers.
• The main difference is that PCR is used instead
  of DNA blot hybridisation to detect a restriction
  site polymorphism.
• A genomic DNA region is amplified by PCR using
  specific primers and those amplified fragments
  are then digested with a diagnostic restriction
  enzyme to reveal the polymorphism.
• RFLP probes can be anonymous clones, CAPS markers
  require sequence information to design the
  specific PCR primers.

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Advantages

1. CAPS markers are co-dominant.
2. Most CAPS genotypes are easily scored and
   interpreted.
3. CAPS markers require only small quantities of
   genomic DNA.

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Random Amplified Polymorphic DNA (RAPD)

• RAPD markers are another type of PCR-based
  markers that have been used for genetic mapping.
• This approach is based on the amplification of
  random DNA segments with single primers of
  arbitrary nucleotide sequence.
• The oligonucleotide (around 10-bp long) is used
  for PCR at low annealing temperatures.
• When the oligonucleotide hybridises to both DNA
  strands at sites within an appropriate distance
  from each other, the

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• DNA region delimited by these two sites will be
  amplified.
• Small nucleotide changes (polymorphism) at one of
  the two sites may prevent hybridisation of the
  oligonucleotide and hence also prevent DNA
  amplification.

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• Typically a RAPD primer will amplify a given
  fragment from line A and not from line B.
• It will thus be impossible to distinguish an
  homozygous individual AA from an heterozygous
  individual AB.
• In other words, RAPDs are dominant markers and
  are thus less efficient than co-dominant markers
  in extracting information from a given F2
  population.
• Another limitation of RAPD markers is that
  because of the low annealing temperatures used,
  the amplification of a given polymorphic band
  seems to be highly sensitive to PCR conditions
  and hence less consistently reproducible in
  different laboratories.

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Advantages

• Random distribution throughout the genome
• The requirement for small amount of DNA (5-20 ng)
• Easy and quick to assay
• The efficiency to generate a large number of
  markers
• Cost-effectiveness!

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Limitations

1. Dominant nature (heterozygous individuals can not
   be separated from dominant homozygous)
2. Sensitivity to changes in reaction conditions,
   which affects the reproducibility of banding
   patterns
3. The results are not easily reproducible between
   laboratories

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Amplified Fragment Length Polymorphism (AFLP)

• AFLP TM is a patented technology developed by
  KeyGene, Wageningen, The Netherlands.
• In this procedure, the genomic DNA is digested by
  two different restriction enzymes, a rare cutter
  and a frequent cutter.
• Double-stranded adapters are then ligated to the
  ends of the restriction fragments.
• The fragments are then amplified by PCR using
  primers that correspond to the adapter and
  restriction site sequences.

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• These primers have additional nucleotides at the
  3' ends extending into the restriction fragments,
  in order to limit the number of fragments that
  will be amplified.
• The AFLP products are detected by labelling one
  of the two primers, and the labelled DNA
  fragments are separated by electrophoresis in
  denaturing polyacrylamide gels (similar to
  sequencing gels).
• Typically, 50 to 100 amplification products are
  detected in a single lane. Polymorphic bands can
  be identified by comparing the amplification
  products derived from two lines.
• Like RAPDs, AFLPs are typically dominant markers.

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• The procedure of AFLP technique is divided into
  three steps
• Digestion of total cellular DNA with one or more
  restriction enzymes and ligation of restriction
  half-site specific adaptors to all restriction
  fragments.
• Selective amplification of some of these
  fragments with two PCR primers that have
  corresponding adaptor and restriction site
  specific sequences.
• Electrophoretic separation of amplicons on a gel
  matrix, followed by visualisation of the band
  pattern.

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Simple Sequence Repeats (SSR) Microsatellites

• Regions of genome where a short (1-4 base) motif
  is repeated many times (can be repeated 10 to 100
  times)

• These microsatellite repeat sequences are usually
  polymorphic in different lines because of
  variations in the number of repeat units.
• These polymorphisms are called SSR, and can be
  conveniently used as co-dominant genetic markers.

• As compared to CAPS markers, SSR offer the
  additional advantage that they do not involve the
  use of restriction endonucleases and thus avoid
  the problems associated with partial digestions.

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• One common example of a microsatellite is a (CA)
  repeat.
• CA nucleotide repeats are very frequent in human
  and other genomes, and present every few thousand
  base pairs.

• Microsatellites developed for particular species
  can often be applied to closely related species,
  but the percentage of loci that successfully
  amplify may decrease with increasing genetic
  distance.

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Single Nucleotide Polymorphisms (SNPs)
Definition of SNP Single Nucleotide
Polymorphism a single base difference in DNA
sequence among individuals.

• (SNP, pronounced snip), is a DNA sequence
  variation occurring when a single nucleotide - A,
  T, C, or G - in the genome (or other shared
  sequence) differs between members of a species
  (or between paired chromosomes in an individual).

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DNA strand 1 differs from DNA strand 2 at a
single base-pair location (a C/T polymorphism).
In this case we say that there are two alleles 
C and T.





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Factors to consider when choosing markers

• Abundance
• Dependent on frequency at which the marker sites
  occur through out the genome.
• Level of polymorphism
• Determined by rate of mutations in a loci e.g.
  charges in a protein, number of repeats in a core
  sequence in microsatellite, substitutions,
  deletions, insertions etc.

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• Locus specificity
• Homology Vs Non homology of bands need to be
  considered.
• Codominance/dominance
• Allows differentiation of homozygotes and
  heterozygotes and therefore determination of
  genotypes and allele frequencies at a loci

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• Reproducibility
• Repeatability of findings over time and space.
• Labor intensity and safety
• RFLPs Vs SSRs.
• Technical demand
• Skills required, equipment needed
• Hybridization gtPCR-based

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• Operational costs
• Chemicals, supplies, visualization techniques
  e.g. labeling, staining methods etc.
• Development costs
• Construction of genomic libraries and development
  of site specific PCR-primers for SSRs and Probes
  for RFLPs, Sequencing is expensive

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• Quantity of DNA required for analysis
• RFLPs 5-10µg, PCR 5-100ng per reaction
• Important when only small tissues are available.
• Amenability to automation
• Increase in sample throughput
• Manually, 100 PCR reactions gt 2hrs to set up
  Robot lt20 min