MAPPING POPULATIONS: for Linkage Mapping of Genetic Markers

2. INTRODUCTION
The demand being placed on food production globally require greater advances in
genetic improvement of our food crops.
 A crucial step towards achieving these advances is the rapid identification of genetic
elements that can be utilized in breeding food crops.
The identification of genes underlying simply inherited traits has been very
successful.
Most traits of biological and economic interest in crop plants are of quantitative
nature, displaying continuous variation and are under polygenic control and
identification of such genes is difficult.
The development of molecular marker technology has renewed interest in genetic
mapping.
RFLP was the first DNA marker to be developed and used in mapping experiment.
The use of DNA markers has allowed construction of dense linkage maps in many
important plant species and has enabled the mapping of the elusive quantitative trait
loci (QTLs).

3. Genetic
resources
Molecular
biology
Crop
improvement

4. Historical Aspects
• In 1910, Morgan provided the first experimental evidence for the chromosomal
theory: he demonstrated that the inheritance pattern of white-eye gene of
Drosophila indicated it to be located on the X chromosome. One year later, in
1911, Morgan described the essential features of linkage between genes.
• In the year 1913, Sturtevant published the first linkage map of Drosophila.
• Subsequently, morphological markers were used to construct linkage maps in
many species.
• Geneticists mounted search for more abundant markers, and protein
polymorphisms were the first molecular variations used to generate linkage
maps.
• The limited number of protein polymorphisms and the environmental influence
on their expression were the major drawbacks, which favored the development
of DNA markers.

5. LINKAGE MAPPING
 Mapping is putting markers (and genes or QTL) in order, indicating
the relative distances among them, and assigning them to their
linkage groups on the basis of their recombination values from all
pair wise combinations.
 A linkage map may be thought of as a ‘roadmap’ of the
chromosomes derived from two different parents (Paterson,1996a)
 Linkage maps indicate the position and relative genetic distances
between markers along chromosomes.
 Construction of linkage maps requires the following:
(1) a suitable marker system,
(2) an appropriate mapping population, and
(3) software for proper analysis of the data.

6. The first step in mapping of markers is to select two genetically divergent parents expected
to differ in lager number of markers and/or trait of interest.
General Procedure for Linkage Mapping Of
Molecular Markers
The selected parents are crossed and a suitable mapping population is developed
The parents are tested with large number of markers to identify polymorphic markers.
All the individuals of the mapping population are screened with the polymorphic marker; this
is called GENOTYPING.
The marker genotype data are analyzed using a suitable software package to estimate
recombination frequencies and genetic distances between marker pairs

7. Group the markers into linkage groups, select the most likely marker order, and prepare
a marker linkage map.
All individuals of the mapping population are evaluated for phenotypic expression of the
trait, this is known as PHENOTYPING.
The trait phenotype and marker genotype data are analyzed using a suitable computer
program to identify the markers governing the target trait.
Estimate the frequency of recombination between the gene and the markers and
ultimately prepare a linkage map.

8. MAPPING POPULATIONS
A POPULATION THAT IS SUITABLE FOR LINKAGE MAPPING OF GENETIC
MARKERS IS KNOWN AS MAPPING POPULATION.
 Generated by crossing two or more genetically diverse lines and handling the
progeny in a definite fashion. They are usually produced from controlled
crosses.
 Mapping populations serve as basic tools needed for the identification of
genomic regions harbouring genes/ QTLs and for estimating the effects of
QTL.

9.  Primary mapping populations are created by hybridization between two homozygous
lines usually having contrasting forms for the traits of interest.
 Secondary mapping populations on the other hand, are developed by crossing two
lines/individuals selected from a mapping population; they are developed mainly for fine
mapping of the genomic region of interest.
 Mortal mapping populations are short lived populations and each individual of the
populations have their own unique identity.
 Immortal mapping populations are the population which can reproduce itself or can be
reproduced without any change in genetic make up of constituent individuals.
 Bi-parental mapping populations are obtained by the crosses between two parents.
 Multi-parental mapping populations involve more than two parents. E.g. MAGIC.
TYPES OF MAPPING POPULATIONS:
1. PRIMARY AND SECONDARY MAPPING
POPULATIONS.
2. MORTAL AND IMMORTAL MAPPING
POPULATIONS.
3. BIPARENTAL AND MULTIPARENTAL MAPPING
POPULATIONS.

10. Selection of parents for developing mapping
populations
 The two lines selected as parents (P1) and (P2), should be
completely homozygous.
 They should differ for as many qualitative and metric traits as
possible.
 The parents should be polymorphic for as many molecular markers
as possible to afford the construction of dense linkage map.
 The parents of mapping populations must have sufficient variation for
the traits of interest at both the DNA sequence and the phenotype
level.

12. F2 POPULATION
 It consist of the progeny produced by selfing/ sib
mating of the F1 individuals from a cross between
the selected parents.
 Homozygous and heterogeneous population.
 Different plants in F2 population differ with each
other.
 Product of a single meiotic cycle i.e. one round of
recombination.
 The ratios expected for dominant and co
dominant markers are 3:1 and 1:2:1 respectively.

13.  Appropriate population for
preliminary mapping of markers
and oligogenes.
 Useful in identifying heterotic
QTLs.
 Requires less time and efforts for
development.
 Linkage established using F2
population is based on one cycle of
meiosis.
 Precise evaluation of quantitative
traits and their effects of G*E
cannot be done.
 F2 populations are ephemeral and
therefore cannot be maintained
beyond one generation.
 Limited use in fine mapping and
QTL mapping

14. F2 derived F3(F2:3) population
 A F2-derived F3 or F2:3 population is obtained by selfing the
F2 individuals for a single generation and harvesting the
seeds from each F2 plant separately so that each F2 plant is
represented as an individual plant progeny.
 F2:3 populations are suitable for mapping of oligogenic traits
controlled by recessive genes and of QTLs .
 Useful for reconstitution of individual F2 genotypes.
 The construction of these populations requires an extra
season than that of F2 populations.
 The genotype and, particularly, phenotype of the F3
population do not strictly correspond to that of the F2
generation due to one more round of segregation,
recombination, and inbreeding.

15. Doubled Haploids:
 Doubled haploid (DH) plants are obtained
by chromosome doubling of haploid plants
usually derived by culture of anthers/pollen
grains produced by F1 plants.
 Seeds from individual DH plants are
harvested separately and maintained as
DH lines in the same way as RILs.
 The expected ratio for the genes as well as
markers in a DH population is 1:1
irrespective of the marker being dominant
or co dominant.
 DHs are similar to F2 in that they both are
products of one meiotic cycle occurring in
F1. But the differ in the frequency of
recombinants.

16. Double Haploids:-
 Completely
homozygous.
 Perpetual/ Immortal.
 Can be evaluated in
replicated yield trials.
 Suitable for mapping of
both qualitative as well
as quantitative traits.
 Instant production of
homozygous lines.
 Recombination from
male side alone is
accounted
 Non-availability of
suitable haploid
production techniques
 Anther culture induced
variation.
 Differential
regeneration response
of parents.

17. Usually the F1 as a rule is
backcrossed to the
recessive parent i.e., the
parent having the
recessive form of the
target trait. Such a
backcross is called
testcross, and is usually
denoted by B2.
Backcross populations
are generated by
crossing F1 plants with
either of the two
parents of the
concerned F1.
BACKCROSS POPULATION
In the case of co-dominant markers, the order of backcross as well as the phase of
linkage is not important when only markers are to be scored.
But, in the case of dominant markers, the order of backcross is extremely important as
they give difference in segregation ratio with difference in phase of linkage.
The backcross populations offer one specific advantage as they can be further utilized
for marker-assisted backcrossing (MABC) for introgression of the target traits as
proposed in the advanced backcross QTL method (Tanksley and Nelson 1996).

18.  The construction of backcross populations, like that of F2:3 populations, requires
one more generation than that of F2 populations.
 Cannot be evaluated in replicated trials, which makes them unsuitable for QTL
mapping.
 They are not perpetual/they are mortal.
 They capture the recombination events of only one parent, i.e., the F1.
 It requires crossing of the F1 plants with the selected parent, which imposes
additional work and may limit the population size in many crop species.

19. Recombinant Inbred Lines(RILs)
 Recombinant inbred lines(RILs) are a set of
homozygous lines produced by sib mating/selfing
of individual F2 plants.
 RILs are also called F2-derived inbred lines or
single seed descent (SSD) lines because they
are derived from F2 populations usually by the
SSD procedure.
 Wherever possible, F2 plants and their progeny
should be selfed, and sib-mating should be
resorted to only when selfing is not feasible for
some reason.

20.  The SSD procedure is followed for
five or more (usually >8) generations,
during which one seed is harvested
from each plant of the F2 and the later
generations and seeds from all the
plants are composited and planted to
raise the next generation.
 At the end of SSD procedure, seeds
from each plant are harvested
separately to obtain as many RILs as
there are individual plants in the SSD
population.
 Homozygous populations.

22.  RILs are immortal.
 Most commonly used for QTL
mapping.
 Suitable for fine mapping
&map saturation.
 RILs are twice as efficient
as an F 2 population.
 Multilocation trials can be
conducted as they are
homozygous lines.
 Require 6-10 seasons to
develop.
 Development difficult in crops
which show non tolerance to
inbreeding depression.
 Only used for Additive and AXA
type of interactions.
 Genetically variable over time
due to mechanical mixture,
mutations etc..
 Smaller confidence interval.
MERITS DEMERITS

23. Immortalized F2 population
 Immortalized F2 populations can be developed by paired crossing of the randomly
chosen RILs derived from a cross in all possible combinations excluding reciprocals.
 The set of RILs used for crossing along with the F1s produced, provide a true
representation of all possible genotype combinations (including the heterozygotes)
expected in the F2 of the cross from which the RILs are derived.
 The RILs can be maintained by selfing and required quantity of F1 seed can be
produced at will by fresh hybridization.
 This population therefore provides an opportunity to map heterotic QTLs and
interaction effects from multilocation data.

24. ADVANTAGES
 Population identical to the conventional F2 population can be produced and
replicated “n” number of times.
 Individual F2 genotypes can be evaluated over the years and locations.
 Eliminates the need of genotyping the immortalized F2s. Their genotype can
be deduced based on their parental RILs genotypes. Thus economizing the
cost of mapping.
 Can be used for mapping all the traits for which the parents of the RILs and
the RILs show contrasting phenotype.
 It is possible to eliminate the additive X dominance(J) and dominance X
dominance (i) effects.

25. Near Isogenic Lines.(NILs)
 Near-isogenic lines (NILs) are
pairs of homozygous lines that are
identical in genotype, except for a
single gene/locus.
 NILs are generally produced by
backcross procedure. But they
can also be produced by selfing.
 The first strategy used for
molecular mapping was based on
NIL itself.

27.  Immortal populations
 Suitable for gene
tagging
 Useful in functional
genomics
 Require
many
generations
to develop
 Not useful for
linkage mapping.
 Linkage drag
MERITS DEMERITS

29. Marker type F2 RILs DHs NILs B1 B2
Codominant 1:2:1 1:1 1:1 1:1 1:1 1:1
Dominant 3:1 1:1 1:1 1:1 1:0 1:1
Segregation ratios at dominant and codominant marker loci in different mapping
populations
Codominant markers: RFLPs, SSRs, CAPSs
Dominant markers: RAPDs, AFLPs, most SCARs, IISRs, SNPs, DArT, SFPs, RAD markers.

30.  Chromosomal segment substitution lines (CSSLs).
 Backcross inbreed lines(BILs)
 Advanced Intercrossed lines.(AILs)
 Interconnected mapping populations.

31. Multi-parental Population (MAGIC)
 Multi-parent Advanced Generation Intercross (MAGIC) populations are a collection of
RILs produced from a complex cross/ outbreed population involving several parental
lines.
 The parental lines may be inbred lines, clones, or individuals selected on the basis of
their origin or use.
 This concept was first used in mice as “heterogeneous stocks” and later extended to
plants by Mackay and Powell (2007), who also proposed the name MAGIC.
 MAGIC populations are perpetual, lack population structure, can be used for both
linkage and association analysis.
 They are an ideal resource for construction of high-density maps.
 Parents of the MAGIC population may be selected to represent a large part of
variation present in the elite germplasm of a crop species.

32. Inbreeding
Advanced intercrosses
Mixing of parents
Founder selection

33. This complex cross is handled as per the SSD procedure to develop the required number of
RILs, which together constitute the MAGIC population.
A simple approach to generate a MAGIC population is to produce a complex cross involving
multiple, typically eight, parental lines and to isolate RILs from this cross.
The 8 parental lines are crossed in pairs to produce four different single crosses, and these
single crosses are crossed in pairs to generate two double crosses
Finally, the two double crosses are mated together to produce an eight-parent complex cross.

35. APPLICATIONS:
 The short term mapping populations like F2, BC, can be a good starting point
in molecular mapping.
 Long term mapping populations like RILs, DHs, NILs, CSSLs, Immortalized
F2, MAGIC can be developed for precision phenotyping of the traits of
importance.
 RILs, NILs, DHs are homozygous lines and can be used for studying of
additive gene actions or additive QTLs.
 Immortalized F2 population, F2 population, BC poulstion can be used for
studying heterotic QTLs.

36. Parent 1 Parent 2
F1
Back crossing Anther Culture
Selfing
F2
Pedigree Method Bulk Method
SSD
RILs
Immortal mapping populations
NILs DHs
Characterization for targettraits Molecular genotyping
Identification of markers linked to target traits MAS
F2 population
Backcross population
X

37. CONCLUSION:-
Development and characterization of mapping populations should become an integral
part of the ongoing breeding programs in important crops.
The role of geneticists and plant breeders become crucial for reaping the full benefits of
molecular plant breeding.
MARKER
MAPPING
POPULATION
SOFTWARE

38. REFERENCES
 Marker- assisted plant breeding: Principles and Practices by BD Singh and AK Singh
 http://bioinformatics.iasri.res.in
 www.ndsu.edu