One of the first genomes to be completely sequence
was that of Arabidopsis thaliana (Thale cress). The
primary reason for sequencing the genome of a
„weed‟ was its small genome size.
Genome size was a limiting factor in sequencing
using BACs during the 1990s.
The five chromosomes of A. thaliana contain DNA
molecules of 115 Mbp and encode 25,498 genes.
Genome Structure & Organization
The centromeres contain
retroelements, transposons, microsatellites and
Gene families are organized into tandem arrays.
Duplicated segments comprise 58 – 60% of the
genome (24 segments of more than 100 kb in size).
This implies evolution from a tetraploid ancestor.
Functional proteins: 69% similar, 30% unknown.
Plant breeders have developed thousands of new
varieties by selection of parental genotypes with
desirable phenotypes followed by controlled
Molecular markers linked to specific phenotypic
traits (Quantitative Trait Loci) are now being applied
to screen for varieties with the desired traits prior to
selection of their genetic material for incorporation
into breeding programs.
Determinate and Indeterminate
In the case where both the parental genotypes
(donor and recipient) are known, the F1 progeny is
designated as ‘determinate’.
When F1 hybrids are „open pollinated‟ in the
field, the resultant F2 generation is designated as
The commercial seed industry is based on the
development of determinate hybrids.
Phenotypic characteristics are designated as „traits‟.
Some traits such as flower color can be observed
physically, whereas others such as „grain
yield‟, „disease tolerance‟ and „herbicide resistance‟
need to be evaluated by subjecting the plant to
DISCRETE and CONTINUOUS.
When a DNA marker can be linked to a specific
trait, it is referred to as a “Quantitative Trait Locus”.
Genomic loci are subject to genetic rearrangement
via the twin processes of recombination and
recombination events may result in two or more loci
that appear to be linked to each other and are
designated to be in „Linkage disequilibrium‟. The
converse of the above phenomenon is „Linkage
When two varieties of a specific crop plant, the „wild
type‟ and „inbred line‟ are crossed to develop a novel
F1 hybrid, the desirable traits from the „wild type‟ are
acquired by the F1 generation, however the process
may also result in the acquisition of some undesired
traits. This phenomenon is known as ‘linkage
drag’. The solution to this problem lies in backcrossing or in genetic modification using horizontal
gene transfer into inbred lines.
EPISTASIS AND ENVIRONMENTAL EFFECTS
Not all F1 hybrids may exhibit the traits contained in
the genetic material inherited from their parental
genomes. This effect can be attributed to the
phenomenon of „Epistasis‟ as well as the influence of
environmental factors on the expression of specific
What are the three forces which drive the evolution
of the genome?
DNA MARKERS, QTL & MAS
QTL Mapping: Linkage Mapping
Marker Assisted Selection
Traits designated as A, B, X and Y segregate
independently of each other.
Green indicated pollen and red indicates ovules.
Marker Assisted Selection (MAS) relies on markers
which are tightly linked to the locus expressing the
desired trait. Ideally, two markers will be more
accurate at predicting the presence or absence of the
trait as compared to a single marker. The distance
between the marker and the trait should not be in
excess of 5 cM.
rA = 5 cM
rB = 4 cM
rA = 5 cM
rB = 4 cM
Locus expressing a
ADVANTAGES OF MAS
Simpler than phenotypic screening especially in the
case of complex traits.
Selection can be carried out at the seedling stage.
Single plants can be selected: both homozygotes and
heterozygotes can be identified.
Reduction in the space required for breeding as only
selected germplasm is propagated.
APPLICATIONS OF MAS
Marker assisted evaluation of breeding material.
Marker assisted backcrossing.
Marker assisted pyramiding.
Early generation MAS.
MARKER ASSISTED EVALUATION
Evaluation of cultivars for purity of breeding
Assessment of genetic diversity and parental
Study of heterosis (hybrid vigour) : do a majority of
hybrids exhibit high levels of genetic polymorphism?
Allelic diversity, rare genotypes.
MARKER ASSISTED BACKCROSSING
DNA markers greatly increase the efficiency of
Useful in the case of screening for traits which are
difficult to detect in the phenotype (e.g.: insect
Backcrossing reduced the level of introgression and
results in a lower degree of linkage drag.
Background selection is used to screen for integrity
of the recurrent parent genome.
Marker Assisted Backcrossing: Schematic
MARKER ASSISTED PYRAMIDING
Marker assisted pyramiding involves breeding
several different varieties in order to develop a
genetically distinct „pedigree‟.
Markers can be developed for specific traits on each
of the varieties being inbred and then applied to
determine the gain of the trait or its subsequent loss
over several cycles of breeding.
for each of
Elite variety should technically exhibit traits from all the 4 parents.
EARLY GENERATION MAS
Early generation MAS facilitates the elimination of
F1 hybrids which do not carry the desired traits as
reflected by their DNA profile.
Single large scale (SLS-MAS) relies on markers
which are less than 5cM on either side of a locus.
The selection of homozygotes or heterozygotes for a
specific locus facilitates the linkage of heterozygosity
screening combined with MAS is
essential because not all traits can be identified using
molecular genetic approaches.
Certain traits may be under the influence of more
than one QTL.
REASONS FOR LOW IMPACT OF MAS
MAS results are not published.
Reliability and accuracy.
Insufficient linkage between marker and QTL.
Limited markers and limited polymorphism.
Effects of genetic background.
QTLs and environmental effects.
High cost of MAS.
MAS ARE NOT PUBLISHED
Commercial plant breeders do not publish MAS data
as it may reveal information related to newly
developed plant varieties.
Although newly developed plant varieties are
protected, release of information relate to DNA
markers may compromise commercial interests.
RELIABILITY AND ACCURACY
Polygenic traits which are linked to more than one
QTL are difficult to establish.
In the case of small populations, sampling bias can
result in loss of accuracy.
A large toolbox of markers is required to establish
QTLs with a high degree of precision.
The loss of linkage may arise from a recombination
event between the markers and the QTL.
Case I: Tightly linked
Case II: Insufficient linkage
LIMITED MARKERS & POLYPMORPHISM
complete genomes of many commercially
cultivated crops are not currently available at public
In cases where genomes are available, there has been
no established QTLs between genotype and
Markers have to be highly polymorphic in order to
serve as tools for linkage analysis.
EFFECT OF GENETIC BACKGROUND
Markers developed in one breeding population may
not be effective in other breeding populations, a
phenomenon which can be attributed to epistatic
interaction between gene products.
In many cases the expression of specific genes is
controlled by environmental cues and QTLs linked to
these genes may be ineffective in determining the
relationship between genotype and phenotype.
HIGH COST OF MAS
The development of MAS based breeding programs
requires a significant investment in the isolation of
molecular markers, testing of these markers as well
as the establishment of inbred lines. These puts
molecular markers beyond the reach of small
There is a lack of knowledge transfer between
scientists at research laboratories and breeding
stations. This may be the result of the need to protect
Intellectual Property (IP).
Research scientists are driven by the need to publish
rather than to assist breeders in long-term field
Fundamental concepts in plant breeding may not be
understood by plant breeders and other plant
Highly specialized equipment for high-throughput
analysis is not available to plant breeders.
Marker assisted backcrossing.
Efficiency of gene pyramiding.
Qualitative improvement through pyramiding.
Polygenic trait improvement by gene pyramiding.
H 1,2,3,4 (Node)
H 1,2,3,4,5,6 (Root genotype)
Gene Pyramiding Scheme
The objective of molecular breeding is to develop plant varieties
with desired traits. Unlike conventional breeding which is founded
on selection of the basis of „Phenotypes only‟, molecular breeding
is based on the genetic engineering of plants with specific genes
which will result in the desired „Phenotype‟.
Molecular Breeding Strategies
Steps in Molecular Breeding
1. Identification of the desired „trait(s)‟.
2. Characterization of the pathway.
3. Identifying genes involved in the pathway.
4. Gene isolation
5. The construct
6. The transformation and delivery system.
8. Screening and commercialization.
Step 1: The trait
Let us consider a hypothetical case in which a protein “DRR” is
linked to drought tolerance in Oryza sativa. This protein is found
only in wild type O. sativa variety WT-6.
Step 2: Pathway Characterization
Protein DRR is not the product of a single gene, rather, it is the
end product of a pathway, the enzymes of which are encoded by
the genes Drase1, Drbase1, Drcase4 and Drrase4.
Step 3: Gene / Gene Cluster
Each of these genes is encoded on a different chromosome in WT-6
Step 4: Gene Isolation
Have you already learnt to isolate the genes? Yes, using PCR.
Step 5: The Construct
The genes encoding each of the enzymes involved in the
biosynthetic pathway are isolated and linked together to form a
single gene construct. A single promoter or multiple promoters
may regulate the expression of the genes.
Step 6: T & D System
We have to develop a suitable vector or transformation process in order to deliver
our genetic construct to its intended target chromosome.
Step 7: Transformation
Transformation can be carried out using Biolistics, Agrobacterim containing Ti
plasmid, CaMV or other viral mediated transformations.
Plant Tissue Culture System
Step 8: Screening / Commercializing
Finally, we screen for transformants carrying our genetic construct
and breed commercial lines based on the molecular breeding
What can go wrong ?
When a „foreign‟ or „engineered gene‟ is introduced into a
plant, the following situations can be encountered:
1. Gene does not integrate into the host genome / expresses
2. Gene integrates but is not expressed.
3. Gene is expressed only in the first generation.
What makes GE difficult?
1. Fate of RNAs.
2. Promoter functionality.
3. Loss of introduced gene(s) as a result of recombination.
4. Lethal introductions.
5. Interference in regulatory pathways.