MOLECULAR BREEDING

PLANT MOLECULAR GENETICS

The Model Plant: Arabidopsis thaliana

The Genome

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

1. The centromeres contain retroelements, transposons, microsatellites and some genes.

2. Gene families are organized into tandem arrays.

3. Duplicated segments comprise 58 – 60% of the genome (24 segments of more than 100 kb in size).

4. This implies evolution from a tetraploid ancestor.

5. Functional proteins: 69% similar, 30% unknown.

Arabidopsis thaliana: Chromosomes

Chloroplast DNA

Large Single Copy (LSC)

Small Single Copy (SSC)

Inverted Repeats (IR)

Mitochondrial DNA

The mitochondrial genome in plant cells is the result of duplication events.

INTRODUCTION TO PLANT GENETICS

The concept of ‘Breeding’.Hybrids: Determinate and Indeterminate.Quantitative Traits.Linkage: equilibrium and disequilibrium.Linkage drag.Gene Stacking.Back-crossing and the recurrent parent.Marker Assisted Selection. Environmental effects and epistasis.

Norman Borlaug

1914 - 2009

INTRODUCTION

Plant breeders have developed thousands of new varieties by selection of parental genotypes with desirable phenotypes followed by controlled breeding. 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 ‘indeterminate’.

The commercial seed industry is based on the development of determinate hybrids.

QUANTITATIVE TRAITS

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 specific challenges.

DISCRETE and CONTINUOUS. When a DNA marker can be linked to a

specific trait, it is referred to as a “Quantitative Trait Locus”.

LINKAGE EQUILIBRIUM

Genomic loci are subject to genetic rearrangement via the twin processes of recombination and transposition. In some cases successive 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 equilibrium’.

LINKAGE DRAG

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 back-crossing 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 genes.

What are the three forces which drive the evolution of the genome?

DNA MARKERS, QTL & MAS

Population Development

QTL Mapping: Linkage Mapping

QTL Validation

Marker Validation

Marker Assisted Selection

A B X Y

A B X Y

Traits designated as A, B, X and Y segregate independently of each other.

Green indicated pollen and red indicates ovules.

A B

X Y

A X

A Y

B X

B Y

F1 CZ

F2 A CA ZX CX Z

A CA ZY CY Z

B CB ZX CX Z

B CB ZY CY Z

Recruit

GENETIC MARKERS

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

Marker Locus expressing a specific trait

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

1. Marker assisted evaluation of breeding material.

2. Marker assisted backcrossing.3. Marker assisted pyramiding.4. Early generation MAS.5. Combined MAS.

MARKER ASSISTED EVALUATION

Evaluation of cultivars for purity of breeding material.

Assessment of genetic diversity and parental selection.

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 selection.

Useful in the case of screening for traits which are difficult to detect in the phenotype (e.g.: insect resistance).

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.

A B

X Y

A X

A Y

B X

B Y

F1 X Y

F2 A XA YXX

X Y

A XA YY Y

X Y

B XB YXX

X Y

B XB YX YY Y

Marker Assisted Backcrossing: Schematic

Recurrent Parent

F0

F1

F2

F3

X Wild Type

X50:50 Recurrent Parent

X75: 25 Recurrent Parent

87.5: 12.5

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.

Elite variety should technically exhibit traits from all the 4 parents.

DNA Markers can be

developed for each of the specific

QTLs and

tracked over

successive generation

s.

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 on fitness.

COMBINED MAS

Phenotypic 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

1. MAS results are not published.2. Reliability and accuracy.3. Insufficient linkage between marker and

QTL.4. Limited markers and limited polymorphism.5. Effects of genetic background.6. QTLs and environmental effects.7. High cost of MAS.8. Application gap.9. Knowledge gap.

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.

INSUFFICIENT LINKAGE

The loss of linkage may arise from a recombination event between the markers and the QTL.

QTL

Gene

Recombination Site

Case I: Tightly linked

Case II: Insufficient linkage

LIMITED MARKERS & POLYPMORPHISM

The complete genomes of many commercially cultivated crops are not currently available at public databases.

In cases where genomes are available, there has been no established QTLs between genotype and phenotype.

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.

ENVIRONMENTAL EFFECTS

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 breeders.

APPLICATION GAP

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 experiments.

KNOWLEDGE GAP

Fundamental concepts in plant breeding may not be understood by plant breeders and other plant scientists.

Highly specialized equipment for high-throughput analysis is not available to plant breeders.

GENE PYRAMIDING

1. Gene pyramiding.2. Marker assisted backcrossing.3. Efficiency of gene pyramiding.4. Qualitative improvement through

pyramiding.5. Polygenic trait improvement by gene

pyramiding.

H1,2 H3,4 H5,6

H 1,2,3,4 (Node)

F0

F1

F2

F3

Gene Pyramiding Scheme

P1 P2 P3 P4 P5 P6FounderParents

H 1,2,3,4,5,6 (Root genotype)

Pedigree

H (1,2,3,4,5,6)(1,2,3,4,5,6) Ideotype

Molecular Breeding

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

Introgression of specific

trait encoding genes

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.

7. Transformation.

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.

DRR

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.

DRa

DRb

DRc

DRd

DRR

Drase1

Drbase1

Drcase4

Drrase4

Step 3: Gene / Gene Cluster

Each of these genes is encoded on a different

chromosome in WT-6

1 3 7 8

Drase1

Drbase1

Drcase4

Drrase4

Step 4: Gene Isolation

Have you already learnt to isolate the genes? Yes,

using PCR.

Drase1

Drbase1

Drcase4

Drrase4

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.

Drase1 Drbase1 Drcase4 Drrase4

Promoter

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.

Inducible promoter

Recombination site

Recombination site

Selectable Marker

Reporter gene

Construct

Step 7: Transformation

Transformation can be carried out using Biolistics,

Agrobacterim containing Ti plasmid, CaMV or other viral

mediated transformations.

Plant Tissue Culture System

Callus transformed using Biolistics

Screening

Regeneration

Shoot regeneration

Root regeneration

Grow-out and seed harvesting

Step 8: Screening / Commercializing

Finally, we screen for transformants carrying our genetic construct and breed commercial lines based on the molecular breeding strategy.

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 transiently.

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.

Transgene Escape

QUESTION BANK

1. What is the meaning of the term ‘linkage drag’ ?

2. If you were to choose between conventional breeding and molecular breeding, which one would you select and why?

Thank You!