This document provides information about genetic linkage and genetic mapping. It discusses: - The history of genetic linkage mapping beginning with Mendel's laws and the work of Morgan and Sturtevant in the early 1900s. - The three main types of genetic maps - linkage maps, cytogenetic maps, and physical maps. Linkage maps show the relative locations of genetic markers based on recombination frequencies. - How recombination frequencies are used to estimate genetic distances and convert them to map distances in centimorgans using functions like Haldane and Kosambi. - Software used for genetic mapping and estimating recombination rates from different mapping populations. - The mapping of quantitative trait loci which

1. .SUBMITED TO
Dr. Kamal Dev
Sharma
Department of
Agriculture
Biotechnology
SUBMITED BY
Ravindra
Kamble
A-2016-30-004
M.Sc. 1st year
1

2.  The tendency of two or more genes or loci being
inherited together is known as linkage.
 A linkage map is schematic representation of the
relative locations of various genetic markers
present in the chromosomes of an organism as
determined from the frequency of recombination
between pairs of markers.
What is linkage
2

3. 3

4. 4

5. HISTORY OF GENETIC LINKAGE
 Mendel’s Laws
 Law of segregation
 Each parent randomly passes one of two alleles to
offspring
 Law of Independent Assortment
 Separate genes for separate traits are passed
independently to offspring
 Traits should appear in offspring in the ratio of 9:3:3:1
o Linkage
 Thomas Hunt Morgan and his student Alfred Sturtevant
found that recombination frequency is a good predictor of
distance between genes
 Genes that are inherited together must be closer to
one another – linked
 Generated the first linkage maps
 Serves as an important basis for understanding genetic
association studies
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6. Model Organisms
Fruit Flies, plants, etc.
Extremely important for
understanding human
genetics Fruit flies can
produce new generations
of 400+ offspring
approximately every week!
Can very quickly
understand the genetics of
trait heritability
 The concept of genetic linkage is known since the studies of Morgan
1911
 Sturtevant published genetic map of Chromosome X of Drossophilla in
1913
 First partial genetic map of maize was published in 1935
by Emerson et. al.
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7. GENETIC MAPPING
 A genetic map is schematic representation of various genetic
marker in the specific order , in which they are located in
chromosome along with the distance between them .
 Genetic map have been constructed by using Three diverse
strategies to generate three different types of maps:
1.Linkage map
2.Cytogenic or cytological map
3.Physical map
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8. LINKAGE MAP
 A linkage map is schematic representation of the relative locations of
various genetic markers present in the chromosomes of an organism as
determined from the frequency of recombination between pairs of
markers.
 The recombination frequencies between marker pairs are estimated
from suitable mapping population and are converted to map or genetic
distance.
 Based on the genetic distance, the marker groups and their order in the
linkage groups is depicted as the linkage maps but there recombination
frequency shows considerable variation in the different region of the
genome and heterochromatic regions like centromeres exhibit
considerably Reduced
recombination frequencies.
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9. CYTOGENIC MAP
 A cytogenic map depicts the location of various genes in the
chromosome of species relative of specific microscopically observable
landmarks in the chromosome.
 In most cases, each chromosome has a characteristics banding pattern,
which may be either naturally present. i.e. Giemsa C.
 Even morphological land mark like centromeres, nucleolus–organizing
regions, knobs, etc. and heritable heterochromatic regions of identifiable
shape have been used for mapping.
 Cytogenic map may use one or more of the following approaches:
1.Fish: including (mcFISH)
2.Human mouse somatic cell hybridization
3. Analysis of small changes in polytene chromosome and
genetic alteration associated with them.
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10. PHYSICAL MAPS
 In a physical map, the gene/molecular marker are depicted in the same
order as they occur in the chromosome but distance between adjacent
gene/markers are depicted in terms of base pairs.
 The distance in the terms of base pairs is known as physical distance
and determined by either hybridization of appropriate probes or
sequence alignment to a good quality reference genome.
Physical mapping usually involves:
1. Cloning of many piece of chromosomal DNA.
2. Characterization of these fragments for size.
3. Determination of there relative locations along the chromosome
using suitable technique like mcFISH. 10

11.  The ultimate physical map of any genome is good quality genome
sequence that is fully annotated to depict all the functional elements of
genome.
 Reasonably good quality genome sequence are available for several
species but their complete and reliable annotation remains to be
accomplished.
11

12. 12

13. SOFTWARE FOR MAPPING OF MOLECULAR
MARKER
 A number of computer programs like QTL Cartographer, Linkage1,
GMendel, MapMaker, MapManager, etc., have been developed for
this purpose.
 A linkage mapping software should be easy to use, have easy data
preparation, provide for application of suitable statistical tools, and
generate easily understandable outputs with facility of graphic
visualization.
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14. ESTIMATION OF RECOMBINATION RATES
 Let us suppose that two genes, viz., a (alleles A and a) and b (alleles B
and b), are linked and two lines with the genotypes AA BB and aa bb
are crossed to produce the F1 Aa Bb.
 This F1 will produce four types of gametes (AB, Ab, aB, and ab) and
testcross progeny (Aa Bb, Aa bb, aa Bb, and aa bb).
 The gametes AB and ab (and the testcross progeny Aa Bb and aa bb)
represent the parental allelic combinations, while the gametes Ab and
aB (and the testcross progeny Aa bb and aa Bb) are the recombinant
types.
 It should be noted that the recombinant types will be produced by one
crossing over event between the genes a and b. 14

15. The frequency of recombination between the genes a and b can be
estimated as follows:
 This estimation of recombination frequency (r) on the basis of
phenotypic data from a testcross population is possible because this
population permits visualization of the gametes produced by the F1
hybrid.
 Some other mapping populations, such as backcross with the recessive
parent and that with the dominant parent and doubled haploid (DH)
populations, also allow visualization of the F1 gametes. Therefore, r can
be estimated from these populations in the same manner as described
above.
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16.  However, in F2 and recombinant inbred line (RIL) and other similar
populations, r cannot be directly estimated.
 In such populations, the maximum likelihood method has to be used
to obtain the most probable estimate of r But in the case of RILs, a
simpler approach for estimation of r is to first calculate R, which is
the proportion of inbred lines, in which the genes a and b have
recombined.
 Then the value of r is estimated from R following Haldane and
Waddington (1931), who showed that R ¼ 2r/(1 + 2r), which leads to
r ¼ R/[2 (1-R)].
 Therefore, when the value of r is very small, the value of R is
approximately 2r.
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17. GENETIC DISTANCE
 The frequency of recombination depends on distance between
the two given genes, it could be used as a measure of the
distance between them and as the basis for linkage mapping.
 However, recombination frequency cannot be directly used as a
measure of the genetic distance for the following reason.
1. When two genes are located close to each other, only a single
crossing over may be expected to take place between them,
and each crossing over would lead to recombination.
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18. 2. In any case, no matter how far apart two genes are located in a
chromosome, the frequency of recombination between them cannot
exceed 50 %, which is the frequency of recombinants obtained with
independent segregation of genes.
 Thus, in general, the correspondence between recombination
frequency and genetic distance progressively declines with the
increasing distance between the linked genes.
 In view of this, recombination frequencies have to be corrected for the
occurrence of multiple crossovers to obtain the estimates of genetic
distance from them.
 There are several methods, called mapping functions, for converting
recombination frequency into genetic distance, but the two most
commonly used methods are those proposed by
Haldane (1919) and Kosambi (1944).
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19.  The Haldane mapping function corrects recombination frequencies
for multiple crossing over events assuming that occurrence of a
crossing over does not affect the likelihood of another crossing over
in the neighboring regions of the chromosome, i.e., there is no
interference.
 Let us suppose that genes A, B, and C are located in the given order
in a chromosome
1. If there were no multiple crossing overs, a recombination between
the genes A and C will be observed whenever there is a
recombination between the genes A and B or the genes B and C.
 Therefore, the frequency of recombination between A and C
(denoted by rAC) will equal the total of recombination frequencies
between A and B (rAB) and between B and C (rBC).
The Haldane Distance
19

20.  Thus,
2. But multiple crossing overs do take place, and they tend to reduce
the recombination rates between the genes.
 In this case, two crossing overs could occur, one between genes A
and B and the other between genes B and C; the frequency of this
event will equal the product of the frequencies of crossing overs
between the two pairs of genes (rAB . rBC).
3. Since the double crossing over event can occur in two different
ways,
• i.e. crossing over between A and B, followed by that between B and
C, and viceversa, the frequency of double crossing over would equal
2rAB ∙ rBC.
 Therefore, the observed frequency of recombination between the
genes A and C will be lower (by 2rAB ∙ rBC) than otherwise expected.
 Thus,
20

21.  The above equation can be rewritten, simplified, transformed to
make the relationships linear, generalized for any number of loci, and
ultimately simplified to yield the Haldane genetic distance (m) in
Morgans as a function of r as follows:
 Since map distances are generally in centimorgans (cM), and one
Morgan comprises 100 cM, the above equation may be written as
follows:
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22. MAPPING OF QUANTITATIVE TRAIT LOCI
 A QTL is defined as “a region of the genome that is associated with an
effect of a quantitative trait.” So a QTL can be a single gene, or it may be
a cluster of linked genes that affect the traits.
 QTL mapping studies have reported in most of the crop plants for
diverse traits like yield, quality, disease and insect pest resistance,
abiotic stress tolerance and environmental adaptation.
 QTL term coined by Gelderman in 1975.
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23. PRINCIPLES OF QTL MAPPING
 QTL analysis is based on the principle of detecting an association
between phenotype and the genotype of markers.
 The markers are used to partition the mapping population in to different
genotypic classes based on genotypes at the marker locus, and apply
the correlative statistics to determine whether the individual of one
genotype differ significantly with the individuals of other genotype with
respect to the trait under study.
 A significant P value obtained for the differences between the marker
and QTL is due to recombination.
 The closer a marker is from a QTL, the lower the chance of
recombination occurring between marker and QTL.
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24.  Therefore, the QTL and marker will be usually be inherited together in
the progeny, and the mean of the group with the tightly-linked marker
will be significantly different (P < 0.05) to the mean of the group without
the marker.
 When a marker is loosely-linked or unlinked to a QTL, there is
independent segregation of the marker and QTL.
 In this situation, there will be no significant difference between means of
the genotype groups based on the presence or absence of the loosely
linked marker.
 Unlinked markers located far apart or on different chromosomes to the
QTL are randomly inherited with the QTL; therefore, no significant
differences between means of the genotype groups will be detected.
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25. STEPS IN QTL MAPPING
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Development of mapping population
Phenotyping
Genotyping
Generating Saturated linkage map
QTL Detection

26. DEVELOPING OF MAPPING POPULATION
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27. PHENOTYPING OF MAPPING POPULATION
 Strictly speaking there should not be any missing data, but limited
amounts of missing data can be tolerated. The missing data in the
population affects the sample size and in turn affect the power of QTL
mapping.
 The data is pooled over location and replication to obtain a single
quantitative value for the line. It is also necessary to measure the target
traits in experiments conducted in multiple location to have better
understanding of the QTL x Environment interaction.
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28. GENOTYPING
 All the individuals/lines of the mapping populations are now
analyzed using these polymorphic marker.
 Two parents of the mapping population are tested with large number
of markers covering the entire genome, and polymorphic markers
are identified.
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29. SATURATED LINKAGE MAP
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30. QTL DETECTION
 The basic purpose of QTL mapping is to detect QTL, while minimizing
the occurrence of false positive .
• i.e. declaring an association between a marker and QTL when in fact it
does not exists.
 The tests for QTL or trait association are often performed by the
following approaches:
a) Single Marker Analysis (SMA)
b) Simple Interval Mapping (SIM)
c) Composite Interval Mapping (CIM)
d) Multiple Interval Mapping (MIM)
e) AB-QTL Analysis 30

31. GENOME WIDE ASSOCIATION MAPPING
 GWA studies
 Aim to find genetic variants that are associated with
traits
 Typically used to elucidate complex disease traits
 Focus on SNPs, Indels, CNVs
 Most often Case/Control Studies
1. SNP (Single Nucleotide Polymorphism)
 Change in a single nucleotide position
2. Indel (Insertion/Deletion)
 Describes the insertion or deletion of nucleotides
3. CNV (Copy number variations)
 Large deletions or duplications of genetic material 31

32. GWA STUDY HISTORY
Human Genome Project (1990-2003)
 Decade long international project to determine the complete human genome
sequence
 Provided the reference genome for future research on genome variation
Human Hap Map (2002-2009)
 Sequencing whole genomes is expensive.
 Needed a shortcut to understand how variation contributes to disease.
 Mapped millions of common known SNPs in 269 individuals.
 Theory that common SNPs are inherited and could be predictive of
associated disease
 Determine how SNPs from case/control studies associate with human disease
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33. ASSOCIATION MAPPING (AM)
 Association mapping, also known as "linkage disequilibrium mapping", is
a method of mapping quantitative trait loci (QTLs) that takes advantage of
linkage disequilibrium to link phenotypes to genotypes.
 Uses the diverse lines from the natural populations or germplasm
collections.
 Discovers linked markers associated to gene controlling the trait.
 Association studies are based on the assumption that a marker locus is
‘sufficiently close’ to a trait locus so that some marker allele would be
‘travelling’ along with the trait allele through many generations during
recombination (Murillo and Greenberg, 2008.)
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34. Major goal
 To identify inter-individual genetic variants, mostly single
nucleotide polymorphisms (SNPs), which show the strongest
association with the phenotype of interest, either because they
are causal or, more likely, statistically correlated or in linkage
disequilibrium (LD) with an unobserved causal variants.
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36. 36

37. Steps in
Association
Mapping
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39. 39

40. ADVANTAGES OF AM OVER LINKAGE MAPPING
1. Much higher mapping resolution,
2. Greater allele number and broader reference population
3. Possibility of exploiting historically measured trait data
4. Less research time in establishing an association
40

41. HIGH-THROUGHPUT SNP GENOTYPING
 High-throughput SNP genotyping offers a number of advantages
over previous marker systems, including an abundance of
markers, rapid processing of large populations, a variety of
genotyping systems to meet different needs, and straightforward
allele calling and database storage due to the bi-allelic nature of
SNP markers.
 On the other hand, routine deployment of trait-specific SNP
markers requires flexible, low-cost systems for genotyping
smaller numbers of SNPs across large breeding populations,
using platforms such as Fluidigm’s Dynamic Arrays™, Douglas
Scientific’s Array Tape™, and LGC’s automated systems for
running KASP™ markers 41

42. EXAMPLES OF HIGH-THROUGHPUT SNP
GENOTYPING
42

43. ADVANTAGES OF SNP GENOTYPING
 The main advantages of SNP markers relate to their ease of data management
along with their flexibility, speed, and cost-effectiveness.
 Bi-allelic SNP markers are straightforward to merge data across groups and
create large databases of marker information, since there are only two alleles
per locus and different genotyping platforms will provide the same allele calls
once proper data has been performed.
 It is important to have a bioinformatics data management and curation team to
convert SNP markers from different platforms to be on the same DNA strand,
that is less challenging than trying to harmonize SSR allele sizes from different
systems. With the help of a high quality reference genome, merging sequence
and SNP data also enables more powerful analyses of the complete SNP
catalog or “SNP universe” for each species. 43

44. CASE STUDIES
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45. CASE STUDIES
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