Association Mapping

And
Its Role In Plant Breeding
Submitted by:
Mahendrakumar N. Chaudhari
Ph.D. 2nd Sem

Submitted to:
Dr. A. Parihar
Associate Professor
Dept. of Plant Molecular Biology and Bio Technology
B. A. C. A., A. A. U, Anand
 Concept of Association Mapping
• Association mapping, also known as "linkage disequilibrium mapping", is a

method of mapping Quantitative Trait Loci (QTLs) that takes advantage of historic

linkage disequilibrium to link phenotypes (observable characteristics)

to genotypes (the genetic constitution of organisms).

• Linkage disequilibrium is the non-random association of alleles at different loci in

a given population. Loci are said to be in linkage disequilibrium when the

frequency of association of their different alleles is higher or lower than what

would be expected if the loci were independent and associated randomly.

• Linkage disequilibrium may exist between alleles at different loci without any

genetic linkage between them and independently of whether or not allele

frequencies are in equilibrium (not changing with time)

 A comparison of association genetics and conventional QTL mapping

Attribute QTL mapping Association genetics

Detection goal QTL mapping Quantitative trait Quantitative trait nucleotide, i.e.,
locus, i.e., wide region within physically as close as possible to
specific pedigrees within which causative sequence(s)
a QTN is located
Resolution of causative trait Low – moderate density linkageHigh – disequilibrium within small
polymorphism maps only required physical regions requiring many
markers
Experimental populations for Defined pedigrees, e.g., Linkage disequilibrium experiments:
detection backcross, F2, RI, three and two unrelated individuals
generation pedigrees/families, (“unstructured” populations), large
half-sib families, etc. numbers of small unrelated families.

Marker discovery costs Moderate Moderate for few traits, high for
many traits
Number of markers required 102 –low 103 105 for small genomes –~109 for
for genome coverage large genomes
New York Oraguzie and Wilcox (2007)
 Association Mapping utilizes
ancestral recombination and natural
genetic diversity within a population
to dissect quantitative traits and is
built on the basis of linkage
disequilibrium concept.
It is used to identify relationships
between genetic markers and
phenotypic traits which relies on
historic, unrecorded sources of
linkage disequilibrium.
Association mapping offers three
advantages,
(i) Increased mapping resolution,
(ii) Reduced research time
(iii)Greater allele number

Comparison of linkage analysis with designed mapping populations and

association mapping with diverse collections.
USA Zhu et al. (2008)
Main driving forces of the current interest in association mapping.
Association mapping harnesses the genetic
diversity of natural populations to potentially
resolve complex trait variation to single genes
or individual nucleotides.

 Population structure and

 Familial relatedness,
 Structured association (SA)
 Genomic control (GC)
 Mixed model approach
 Principal component approach

Sequencing,
Genotyping (SNP),
gene expression profiling,
comparative genomics, bioinformatics,
linkage analysis, mutagenesis, and biochemistry

USA Zhu et al. (2008)

 Procedure of Association mapping
Landraces
Cultivars
Choosing a germplasm group with
Farmers breeding material
global genetic diversity
Independently derived varieties
Extant varieties
Core collections

Phenotypic
measurements in the Genotyping with Quantification of LD
multiple replication trials molecular markers using the molecular
and different (e. g., SSRs and SNPs) marker data
environments

Measurements of
Marker-trait correlation with appropriate
population
approach (e. g., GLM, MLM)
characteristics
(structure and
relatedness)

Identification of marker tags associated with

a trait of interest

Cloning and annotation of tagged loci for

potential biological function

Uzbekistan Abdurakhmonov and Abdukarimov (2008)

 Outline for Association Mapping

USA Zhu et al. (2008)

 The functional SNP responsible for variation in
berry number in grapevine is in gray and is not
genotyped.
The genotyped SNPs lie on either side of the
functional SNP.
The genotyped SNP to the right is in high LD
with the functional SNP, while the genotyped
SNP to the left is not in LD with the functional
SNP.
The results of a simple association test (Pearson
correlation) are shown in the bottom box.
The C allele of the high LD SNP is
significantly associated with berry number (P =
0.037), while there is no significant association
for the low LD SNP (P = 0.77).

A Fictional Depiction of a Simple Genotype-Phenotype Association Test.

a. Linkage disequilibrium b. Association mapping
 Locus 1 and Locus 2 present an unusual  Locus 1 and Locus 2 are in LD
pattern of association between alleles A- significant covariance with seed
G and T-C, which deviate from Hardy- colour phenotype is considered
Weinberg expectations, but without any evidence of association.
statistical correlation with a phenotype

China Braulio et al. (2014)

 Comparison of mapping resolution between linkage mapping and AM.

China Braulio et al. (2014)

 LD Quantification (D and r2 ) LD Decay
• D: difference between observed gametic  LD tends to decay with genetic distance
frequencies of haplotypes and expected gametic between the loci under consideration
haplotype (specific pattern and order of alleles that  LD decays by one-half with each generation of
tend to be conserved from generation to random mating.
generation) frequencies under linkage equilibrium.
 In old populations LD is limited to small
• D = PAB − PAPB = (PABPab − PAbPaB) distances.
 LD more quickly declines in outcrossing plant
• Informative for comparisons of different allele species than highly self-pollinating plants.
frequencies across loci and strongly inflated in
a small sample size and low-allele
frequencies.

• r2 : square of correlation coefficient between

two loci; affected by mutation and
recombination. r2 value of equal to 0.1 (10%)
or above consider significant.

Figure: LD decay showing relationship

between genetic distance (cM)
and r2
Zhu et al. (2008)
 Types of Association Mapping

Genome wide association mapping Candidate gene association mapping

(GWAS) (CGAS)

• Search whole genome for causal  Based on available results in

genetic variation. model and non-model plant species
• Doesn’t require any prior information from genetic, biochemical,
on the candidate genes Large physiology studies
number of markers are tested for  Requires identification of SNPs
association with various complex between lines within specific genes.
traits

Merits of Association mapping

 Broader genetic variations with wider background for marker-trait correlations

 Higher resolution mapping
 No need for development of expensive and tedious bi-parental populations

12
Software

Braulio et al. (2014)

Association mapping studies in plants.

Braulio et al. (2014)

Association Mapping of Yield and Yield related Traits Under
Reproductive Stage Drought Stress in Rice (Oryza sativa L.)

• Background:
 The identification and introgression of major-effect QTLs for
grain yield under drought are some of the best and well-proven
approaches for improving the drought tolerance of rice
varieties.
• Objectives of study:
 Screening Malaysian rice germplasm for drought tolerance,
determining the population structure, doing association
mapping of yield and yield-related traits under drought stress
and non-stress (NS) conditions, and carrying out reference
genome-based analysis of qDTY physical regions.

IRRI, Manila, Philippines Swamy et al. (2017)

• Methodology:
• Plants Materials:75 rice genotypes
 Seed are collected from Rice Gene Bank of MARDI, Seberang
Perai, Malaysia.
 The material consisted of Malaysian landraces, breeding lines,
varieties, cultivars and introductions. Six drought tolerant checks
(Vandana, Apo, PSBRC-82, UPLRi7, Mokwoo and IR77298-14-1-
2-10) and two drought susceptible checks (IR64 and MTU1010)
were included in the experiment.
• Genotyping: Molecular work was conducted in the Genotyping
Services Laboratory (GSL) of the Plant Breeding Division at IRRI.
• DNA Extraction and PCR Amplification: Modified CTABMurray
and Thompson (1980)
• Marker Analysis: We used 119 highly polymorphic SSR markers for
genotyping all 75 genotypes.

Swamy et al. (2017)

• Phenotyping under Reproductive Stage (RS) drought stress
and Non-Stress (NS)- Drought was imposed at 30 days after
transplanting (DAT) by draining the water in the field and
withholding it until soil moisture tension reached -70 kPa at
0.2-m depth.
• Phenotypic data recorded for days to 50% flowering (DTF),
plant height (PH) and grain yield (GY).
• Association Analysis
 Allelic Diversity and Cluster Analysis- PowerMarker (v3.25)
 Population Structure- STRUCTURE 2.2 (Pritchard et al.
2000).
 Association Mapping- TASSEL2.1.
 In Silico Analysis of qDTY Regions- were mapped to the
IRGSP 1.0 genome by aligning the SSR primer sequences
using BLAT (Kent, 2002)

Swamy et al. (2017)

Swamy et al. (2017)
Swamy et al. (2017)
• Result
 Yield reduction of up to 60% due to drought and heritability
for grain yield under drought was up to 78%.
 Most of marker trait associations (MTA) identified were on
chromosomes 2, 5, 10, 11 and 12, and their phenotypic
variance (PV) varied from 5% to 19%.
 The important genes families with a high frequency of
presence in the meta-QTLs were F-box domain proteins,
MADS-box proteins, tetra- and pentatricopeptide proteins, no
apical meristem proteins (NAM), heat shock proteins (HSPs),
cytochrome P450 family, ethylene response factors (AP2/
ERFs), auxin response factors (ARFs), brassinosteroids,
CaMK, GDSL-like lipases, membrane proteins, hydrolases,
kinases, peroxidases, peptidase and dismutase family proteins,
zinc finger proteins (ZFPs), leucine zipper motifs (ZIP) and
myeloblastosis (MYB) transcription factors..

Swamy et al. (2017)

 Genome-wide association mapping of vitamins B1 and B2 in
common wheat
• Objective of study: Thiamin deficiency is associated with neurological
problems, including Alzheimer's disease, cognitive deficit and
encephalopathy.
• Riboflavin deficiency destroys mucosal membranes in the digestive
system and can lead to cardiovascular disease and colorectal cancer.
• The aim was to identify loci associated with vitamins B1 and B2 for
quality improvement in bread wheat.
• Methodology: A collection of 166 bread wheat cultivars and advanced
lines from the Yellow and Huai Valley Facultative Region and foreign
countries was used for the study.
• Genotyping and quality control: Genomic DNA was extracted by a
modified method and samples were sent to CapitalBio Corporation for
genotyping with the high-density illumina 90 K infinium SNP array.
• PowerMarker V3.2.5 - Gene diversity, minor allele frequency (MAF)
and polymorphism information content (PIC).
Beijing, China Jieyun Li et al. (2017)
• A high performance liquid chromatography (HPLC) system
and an RF10Axl fluorescence detector was used to determine
vitamins B1 and B2 contents.
• Analysis of variance (ANOVA) -SAS software version 9.2
(SAS Institute Inc., Cary, NC, USA).
• Population structure analysis- Structure v. 2.3.4
• Association analysis- TASSEL software version 5.0 using the
mixed linear model (MLM)

Jieyun Li et al. (2017)

D genome – Lower marker density
6 AS loci is stable
Total 17 loci for B1

Jieyun Li et al. (2017)

Jieyun Li et al. (2017)
 Limitation and Solution
• GWAS offers you very fine resolution, however, the power for detecting a
QTL will be determined by the frequency of alleles, for instance, you may
lose power for detecting rare alleles.
• Another disadvantage of GWAS is that is sensible to the population structure
that may lead to many false positives. There are ways to correct for
population structure.
• However, if your population structure contribute to the variation in your
trait correcting for population structure may lead you to many false
negatives.
• There are several methods that try to bridge the advantages of QTL mapping
and GWAS.
• For examples, MAGIC and NAM populations they have a broader genetic
diversity, and higher resolutions than bi-parental populations and the
frequencies of alleles are quite well represented to reduce the problems of
rare alleles. The development of these types of populations is labor and cost
intensive.
 What is the goal of your study ?

 To identify the causal gene or mutation (GWAS better than QTL mapping) or just
looking for a linked marker (Probably a QTL mapping would do the job).
 Are you interested in the whole genetic architecture of the trait, that is how many
genes and their effect, (GWAS better over QTL mapping) or you are interested in
major QTLs for practical purposes (QTL mapping or GWAS would do the job).
 Previous knowledge of the genetic architecture and heritability of the trait can help
you to make a decision as well.
 IF your trait or traits of interest is underlined by few genes of large effect then
GWAS will be suitable
 If your trait is governed by rare alleles, then QTL mapping would be better or you
would need large populations to detect them with GWAS.
 Future thrust
• Focus on Insect resistance
• Early flowering traits
• Efficient Genotyping and Phenotyping facilities
• Bio informatics tools reduce error
Thank You....