Transcript Ch10planttransformation

Chapter 9: Genetic linkage and maps in
breeding applications
BSA for finding a linked marker
Construction of genetic linkage maps and
tagging economically important genes
Introgression
QTL-analysis
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Genetic linkage
2nd law of Mendel: independent
segregation of 2 loci.
Is valid for loci on different
chromosomes or far apart on the
same chromosome.
Two loci closely together are
physically and genetically linked,
i.e. they segregate together.
Recombination can unlink 2 loci
by a cross-over
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Genetic linkage
• The recombination frequency depends on the
distance on the chromosome.
• The distance along a genetic map is measured in
terms of the frequency of recombination between
genetic markers. This is called the genetic distance
(different from the genetic distance in Chapter 8).
• 1 cM (centiMorgan) corresponds to 1 recombinant in
100 (1% recombination).
• To analyse linkage of genes or markers, a ‘mapping
population’ is needed. This is a progeny from a cross
between parents that are different enough to have
many polymorphisms segregating.
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Genetic linkage
F2
population
AA x BB
Backcross
population
AA x BB
AB
AB x AA
AA AB BB
Mapping populations
BC1
AB AA
Cross pollinator
AB x CD
AC AD BC BD
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Genetic linkage
• The probability of double recombination (same result
for 2 markers as no recombination) is proportional to the
square of the recombination distance between two loci.
• Formulas for the estimation of recombination distance
(bv. Kosambi, Haldane) adjust for the possibility of
double recombination.
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
Genetic linkage
The precision with which genetic distance is measured, is
directly related to the number of individuals which is
studied (if no recombinants found a sample of 20 progeny
plants => recombination fraction = 0; but analyzing 80
additional individuals 1 recombinant can appear =>
recombination fraction = 1).
Typically a primary genetic map is constructed based on
50-100 individuals, permitting to detect recombination
between markers 1-3 cM apart.
If higher precision is required, more individuals should be
analysed
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
Genetic linkage
Creation of a Linkage map + mapping of the trait
• The chromosomal location of a ‘phenotype’ or a
‘mutation’ is determined by identifying nearby
genetic markers which are co-transmitted from
parent to progeny with the phenotype
• Requires extensive phenotyping and genotyping
of many plants of a mapping (=segregating)
population
• Typical experiment: parents and >100 offspring
plants + hundreds to thousands of molecular
markers
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F1 hybrid
Donor parent
Genetic linkage
Recurrent parent

Backcross (F1 x recurrent parent) progeny :
3 SSR markers + 1 phenotypic trait
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2
3
4 5
6 7
8
9
10 11 12 13 14 15 16 17 18 19 20
A
C
D
Number of recombinants:
AC = 6/20 (1, 3, 10, 13, 18, 19)
AD = 4/20 (1, 3, 10, 13)
CD = 2/20
C = 1/20 (plant number 18)
D = 1/20 (plant number 19)
A
4/20
D
C
2/20
6/20
Adapted from Paterson (1996).
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
Genetic linkage
Creation of a Linkage map + mapping of the trait
• Main disadvantage: large progenies and many
DNA-markers are required => time-consuming
and expensive
• For some applications, not necessary to know
the location of the trait (or linked marker) on a
linkage map
=> Alternative approach developed (Bulk
segregant analysis = BSA)
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
BSA
Bulk Segregant Analysis - Definition
• Bulk: it makes use of bulked samples
=> saves time and money
• Segregant: it makes use of a segregating
population
=> but it does not require map information!!!
• Analysis: it screens the whole genome
• Typical experiment: parents and 2 bulks +
hundreds to thousands of molecular markers
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



BSA
BSA compares 2 pooled DNA samples of individuals from
a segregating population originating from a single cross
Within each bulk the individuals are identical for the locus
of interest but are arbitrary for all other loci
Markers polymorphic between the pools are markers
putatively linked to the locus involved in the trait of
interest Parents
Bulks
Segregating population
R
S
R
R
R
S
S
S
R
S
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
BSA
Target: AA
Genotype
bulk
At a locus linked to the trait, using dominant markers
Aa
aA
Aa
Aa
AA
Sample of F2 individuals with
dominant phenotype harboring
the selected locus:
this bulk is made using only
phenotypic information and
homozygote dominants cannot
be distinguished from
heterozygotes
aa
aa
aa
aa
aa
Sample of F2 individuals with
recessive phenotype harboring
the selected locus:
only the homozygote recessive
will be included in this bulk,
made using only phenotypic
information
aa
Genotype
bulk
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BSA
In this scheme
the resistance (R)
gene is linked to
an RAPD marker.
The susceptible
bulk does not
have this marker
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
BSA
Bulk Segregant Analysis – How to set-up an
experiment
1)
2)
3)
4)
5)
Create a segregating population from a single
cross (for example, F1 or BC progeny)
Phenotype the progeny and identify individuals
with extreme trait-phenotypes
Construct DNA bulks of the individuals
displaying the most extreme trait-phenotypes
Genotype the parents and the bulks using
hundreds to thousands of DNA-markers
Identify those markers which distinguish the
bulks and the parents
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
BSA
example
The first published example of BSA =
identification of downy mildew genes in lettuce:
Michelmore et al. (1991) Identification of markers linked to diseaseresistance genes by bulked segregant analysis: a rapid method to
detect markers in specific genomic regions by using segregating
populations.
Proc Nat Acad Sci USA 88: 9828-9832.
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BSA
example
1 2 3 4 5 6 7
Ninh Thuan Phd:
molecular mapping
of blast resistance
genes in a
traditional
Vietnamese rice
cultivar (Chiembac)
1-7 are different
AFLP- primer
combinations,
each time tested
on the parents and
the bulks
A band from
PC6 (primer
combination 6)
is linked to the
resistance
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SCAR
• This marker PC6 could be used to select rice plants at
the seedling stage for resistance, without the need for an
infection test.
• However, an AFLP or RAPD are not very practical to use
for this purpose (AFLP is complex, RAPD not very
reproducible). Therefore, these kind of markers are often
converted in to a simple PCR marker, called a SCAR
sequence characterised amplified region.
• This is done by sequencing the DNA band and designing
2 primers to amplify a specific region of it that will reveal
the polymorphism.
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Genetic map
• In some cases it may be interesting to
localise markers on a genetic map: for
example 2 AFLP markers linked to rice
blast resistance were mapped to
chromosome 12 with the help of STR
markers that were already on the
genetic map (the AFLP markers were
genetically linked to those STR).
• Knowing the map position may help in
cloning the gene or to search for more
closely linked markers in the
neighbourhood
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Genetic map
• Software to produce linkage maps
– Joinmap
– Mapmaker
• Example of website that collects
information on genome and genetic
research, including genetic maps: on corn
www.maizegdb.org
–…
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Genetic map
Example: 1. Creation of mapping population
Resistant AB
Susceptible CD
F1 population
(ac; ad; bc; bd)
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
Genetic map
2. detection of polymorphic loci
SSR markers
size std
19480
19479
19478
19477
19479
R
S
size std
STS markers (RGA
marker)
700
600
500
A
B
400
AFLP markers
300
RFLP markers
F1 individuals
probe A8
R
S
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Genetic map
3. Analysis of segregation
of markers
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
Genetic map
3. Analysis of segregation of markers
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
Genetic map
4. Selection of linkage groups
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
Genetic map
5. Map construction –
genetic distances
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Genetic map
Use of Linkage Maps
• Visualize genomic organisation
• Fine mapping of genes
• Map based cloning
• QTL analysis
• Comparison of maps
– Maps of same species
– Maps of related species (comparative mapping)
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Introgression
In a backcross it is important to have as much of the
recurrent genome as fast as possible to speed up the
breeding process (less generation times). Molecular
markers can be used to screen for this while selecting for
the desirable characteristic of the introgression parent.
In this way 2 BCs + markers will be better than 4 BCs28
Introgression
Molecular markers are especially interesting to avoid
linkage drag : linkage of undesirable characteristics linked
to the desired trait. In a first back cross about 300 plants are
genotyped to look for a cross-over as close as possible on
one side of the desired trait. This plant is then used for the
second backcross. In this way 2 back-crosses with
molecular analysis are better than 100 random backcrosses.
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
QTL
Quantitative trait
– Multiple genes affect the expression of the trait
– The expression in the population is a ‘bell-shaped’ curve: there are many
genotypes and there are no clear phenotypic differences among them:
Frequency distribution
If we want to find DNAmarkers that can help us to
predict this phenotype, we
are searching for several
genetic loci simultaneously
Height
There exist standard
experimental approaches
for this (e.g. QTL analysis)
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
QTL
• A QTL is the location of a gene (or set of genes) that affects
a trait that is measured on a quantitative scale. Examples of
quantitative traits are plant height or grain yield.
• These traits are typically affected by more than one gene,
and also by the environment
• Mapping QTL is not as simple as mapping a single gene that
affects a qualitative because it involves the simultaneous
identification of the chromosomal locations of all the
genetic factors affecting the trait
• Tools required:
–Many polymorphic molecular markers ordered in a linkage map
–Detailed and accurate phenotypic data of the segregating population
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–Appropriate statistical tools
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QTL
Statistical methods for the detection of QTLs
• Single marker analysis
– Tests for differences in the means of the genetic
marker classes: for every polymorphic (-/+) marker
the mean of all the – plants is compared with the
mean of all the + plants. If these means are
different, this indicates linkage of the marker to a
gene that has an effect on the quantitative trait.
– Rough estimation of the location of a QTL
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
QTL
Statistical methods for the detection of QTLs
•
Single marker analysis
 Robust to violations of normality in phenotypic data

• The order of the Marker and the QTL on the genetic
map remains unknown
• Low power if the markers are far apart
• Large progenies required
• It is not possible to distinguish between size of a
QTL effect and it position (relative to the marker): a
marker close to a QTL of small effect will give the
same ‘signal’ as a marker some distance from a QTL
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of large effect

QTL
Statistical methods for the detection of QTLs
• Interval mapping (requires linkage map)
– Estimates the position of a QTL between two
markers
– Systematically searches the genome by calculating a
test statistic at each position of the genome
– Originally based on the maximum likelihood
estimates
– Intervals between adjacent markers along a
chromosome are scanned and the LOD of there
being one QTL versus no QTL at a particular point is
estimated
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–  and : see further.
QTL
• LOD-score = logarithm of the odds =
Logarithm probability together due to linkage
probability together due to chance
• For a good LOD score (>3) it is necessary to have
data from a large progeny and many markers.
• The higher the value of the LOD score, the more
likely the data are if there was a QTL present
compared to the situation when there is no QTL
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QTL
A LOD-profile is constructed along the chromosome,
and the maxima in this profile which exceed a specified
significance level, indicate likely sites of a QTL
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
QTL
Statistical methods for the detection of QTLs
• Interval mapping (requires linkage map)
 • By considering several markers simultaneously, it
allows accurate estimation of QTL-positions
• It is possible to separate the distance and the size of
the gene effect
 The effect of other QTLs present in the genome is
neglected, and only the QTLs with the biggest
phenotypic QTL on a chromosome, estimated
positions and effect may be biased
 Multiple QTL mapping (MQM)
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QTL
Example: analysis QTL
characteristics in tomato
in relation with RFLP
markers:
- Fruit mass
- pH
- Concentration of solids
Displayed via LOD over
the 12 chromosomes
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QTL
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QTL
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QTL
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Overview different marker applications
• Identification individual/cultivar: multilocus SSR
(or AFLP)
• Analysis relationship:
- Sequence if between genera or species
- AFLP within genus or species
- Multilocus SSR within species
• Overview genome: AFLP, multilocus SSR or SNP
• Specific marker for diagnosis or for marker
assisted breeding SCAR or SNP-detectie
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