Plant breeding Methods and use of classical plant breeding. Molecular marker

technology, Marker assisted selection in plant breeding. QTL (Quantitative Trait Loci), Genetic analysis and characterization of crops with various DNA markers and isozymes. Application of Biotechnology in

plant breeding programs., Testing GM crops

Mitesh Shrestha

Plant breeding is the process by which humans change certain aspects of plants over time in order to introduce desired characteristics

Plant Breeding Concept

Increase crop productivity

Domestication

Plant Breeding activities began at least 10.000 years ago in the Fertile Crescent with plant domestication

Challenges: transition from nomadic to a sedentary lifestyle

Increase plant yield

Increase number of edible plants (reduce toxicity)

Landmarks in Plant Breeding

1694 1866 1953

Camerarius crossing as a method to obtain new plant

types

Mendel Empirical evidence

on heredity

Watson, Crick, Wilkins &

Rosalind Franklin model for DNA

structure

1923

Wallace First commercial

hybrid corn

“The Green Revolution” (1960)

Norman Borlaug

Challenge: improve wheat and maize to meet the production needs of developing countries

High yielding semi-dwarf, lodging resistant wheat varieties

Plant Breeding Methods

Conventional breeding

• Mutation or crossing to introduce variability

• Selection based on morphological characteres

• Growth of selected seeds

Challenge: reduce the time needed to complete a breeding program

Objective of plant breeding

• Aims to improve the characteristics of plant so that they become more desirable agronomically and economically.

• Higher yield

• Improved quality

• Disease and insect resistance

• Change in maturity duration

• Agronomic characteristics

Classic/ traditional tools

• Emasculation

• Hybidization

• Wide crossing

• Selection

• Chromosome counting

• Chromosome doubling

• Male sterility

• Triploidy

• Linkage analysis

• Statistical tools

Quantitative trait locus

• Section of DNA (the locus) that correlates with variation in a phenotype (the quantitative trait).

• Linked to, or contains, the genes that control that phenotype.

• Mapped by identifying which molecular markers (such as SNPs or AFLPs) correlate with an observed trait.

• Early step in identifying and sequencing the actual genes that cause the trait variation.

• Quantitative traits are phenotypes (characteristics) that vary in degree and can be attributed to polygenic effects, i.e., the product of two or more genes, and their environment.

Methods of plant breeding

• Self pollinated crop

• Mass selection

• Pure line selection

• Pedigree selection

• Bulk method

• Backcross method

• Single seed Descent and recurrent selection

Mass Selection

• In mass selection, a large number of plants of similar phenotype are selected and their seeds are mixed together to constitute the new variety.

• The plants are selected on the basis of their appearance or phenotype.

• The selection is done for easily observable characters like plant height, grain color, grain size.

Merit of mass selection

• Since a large number of plants are selected the adaptation of original variety is not change

• Often extensive and prolonged yield trials are not necessary. This reduces time and cost needed for developing variety.

• It is less demanding method. Therefore the breeder can devote more time to other breeding programs.

Demerits of Mass Selection

• The variety developed through mass selection show variation and are not as uniform as pureline varieties

• Varieties developed by mass selection are more difficult to identify than pureline in seed certification program.

• Mass selection utilizes the variability already present in a variety or population. Therefore only those varieties/population that show genetic variation can be improved through mass selection. Thus mass selection is limited by the fact that it can not generate variability.

Pureline selection

• Pureline is the progeny of a single, homozygous, self pollinated plant. In pureline selection a large number of plants are selected from a self pollinated crop and are harvested individually.

• Individual plant progenies from them are evaluated and the best progeny is released as a pureline variety.

Advantages of pureline selection

• Pureline selection achieves the maximum possible improvement over the original variety. This is because the variety is the best pureline present in the population.

• Pureline variety are extremely uniform since all the plants in the variety have the same genotype

• Due to its extreme uniformity, the variety is easily identified in seed certification

Disadvantages of pureline selection

• The breeder has to devote more time to pureline selection than to mass selection. This leaves less time for other breeding program.

• The variety developed through pureline selection generally do not have wide adaptation and stability in production possessed by local varieties from which they are developed.

Pedigree selection

• In pedigree method, individual plants are selected from F2 and the subsequent segregating generations and their progenies are tested.

• During the entire operation, a record of all the parent-offspring relationship is kept. This is known as pedigree record.

• Individually plant selection is continued till the progenies become virtually homozygous and they show no segregation, at this stage selection is done among the progenies because there would be no genetic variation within the progenies.

Merits of pedigree method

• This method gives the maximum opportunity for the breeder to use his skill and judgment for the selection of plants particularly in the early segregating generation.

• It is well suited for the improvement of characters which can be easily identified and are simply inherited

• It takes less time than the bulk method to develop a new variety

Demerit of pedigree method

• Maintenance of accurate records takes up valuable time. Sometimes it may be a limiting factor in large breeding program.

• Selection among and within a large number of progenies in every generation is laborious and time consuming.

• The success of this method largely depends upon the skill of the breeder.

Bulk method

• In the bulk method, F2 and the subsequent generations are harvested in mass or as bulks to raise the next generation.

• At the end of bulking period, individual plants are selected and evaluated in a similar manner as in the pedigree method of breeding.

• The duration of bulking may vary from 6-7 or more generations

Merit of bulk method

• The bulk method is simple, convenient and inexpensive

• Little work and attention is needed in F2 and subsequent generations

• No pedigree record is to be kept which saves time and labour

• Artificial selection may be practiced to increase the frequency of desirable types

Demerit of bulk method

• It takes a much longer time to develop a new variety.

• Information on the inheritance of characters cannot be obtained which is often available from the pedigree method

• In some cases at least natural selection may act against the agronomical desirable types

Backcross method

• A cross between a hybrid(F1 or segregating generation) and one of its parents is known as backcross.

• In this method the hybrid and the progenies in the subsequent generation are repeatedly backcrossed to one of their parents.

Merit of backcross method

• The genotype of new variety is nearly identical with of the recurrent parent except for the genes transferred.

• It is not necessary to test the variety developed by the backcross method in extensive yield tests because the performance of the recurrent parent is already known.

• Much smaller populations are needed in the backcross than in the case of pedigree method

Demerit of backcross method

• The new variety generally cannot be superior to the recurrent parent except for the character that is transferred

• Undesirable genes closely linked with the gene being transferred may also be transmitted to the new variety.

• Hybridization has to be done for each backcross. This is often difficult, time taking and costly

Cross pollinated crop

• Population improvement

• Hybrid and synthetic varieties development

• In case of Population improvement, mass selection or its modifications are used to increase the frequency of desirable alleles, thus improving the characteristics of population.

• In case of hybrid and synthetic varieties a variable number of strains are crossed to produce a hybrid population.

• The strains that are crossed are selected on the basis of their combining ability.

Plant Breeding Approach

Classic Breeding

Main Street

Molecular

breeding

Abiotic and biotic resistance breeding

(disease/pest resistance, drought and salt tolerance)

Parent selection and progeny testing Marker-assisted selection (MAS) Genome-wide selection (GWS) Marker-assisted backcross breeding (MABB) QTL-based and genome-wide predictive breeding

P1 x P2 F1 F8-10 F6-7 F4-5 F3 F2

Cultivar

variety

Release

Parent selection Predictive breeding

True/false,

self testing

MAS

for simple traits

Preliminary

Final Yield Test

P1

x

BC1F1

Backcross breeding

MAS

for quantitative traits

Genotyping by sequencing (GBS) RAD-seq and RNA-seq SNP discovery and validation QTL mapping and association analysis Candidate gene identified and clone

Comparative of average physical distance and locus distance in different organisms

Species Genome size (kb) Genetic distance (cM) kb / cM

Phage T4 1.6×102 800 0.2

E. coli 4.2×103 1,750 2.4

Yeast 2.0×104 4,200 4.8

Fungus 2.7×104 1,000 27.0

Nematode 8.0×104 320 250.0

Drosophila 1.4×105 280 500.0

Rice 4.5×105 1,500 300.0

Mouse 3.0×106 1,700 1,800.0

Human race 3.3×106 3,300 1,000.0

Maize 2.5×106 2,500 1,000.0

Needed marker number to reach specific saturated genetic map

Species Human race Rice Maize Arabidopsis Tomato

Genome size

(kb)

(cM)

kb/cM

3.3×106

3300

1000

4.5×105

1500

300

2.5×106

2500

1000

7.0×104

500

140

7.1×105

1500

473

Map saturation

20cM

10cM

5cM

1cM

0.5cM

165

330

660

3300

6600

75

150

300

1500

3000

125

250

500

2500

5000

25

50

100

500

1000

75

150

300

1500

3000

Introduction

Characterization using molecular markers • Molecular characterization is the description of an accession

using molecular markers. • Molecular makers are readily detectable sequence of DNA or

proteins whose inheritance can be monitored. • There are several methods that can be employed in molecular

characterization ,which differ from each other in term of ease of analysis, reproducibility used techniques and their advantages and disadvantages are presented below.

Types of Marker

• The development of genetic marker

– Morphologic marker (eg. flower color, plant height etc.)

– Protein marker / Biochemical marker (eg. isozyme)

– DNA marker / Molecular marker (RFLP, RAPD, SSR etc.)

• Molecular nature of naturally occurred polymorphism

– Point mutation

– Insertion / deletion

– DNA rearrangement

•Some regions of genome are significantly more polymorphic than singly copy sequences Tandem repeats Synteny

•In the use of molecular marker, an important observation is the finding that many distantly related species have co-linear maps for portions of their genomes. Solanaceae Gramineae

Locus & allele Allele frequency & heterozygosity Dominant & co-dominant

Application of Molecular Marker

• Phylogeny

• Genetic diversity

• Molecular Mapping

• Gene tagging

• MAS, marker assisted selection

• Genebank management: duplicate identification

• Fingerprinting

• Quality testing

Classification of Molecular Marker by Detection Technology

• Based on DNA-DNA hybridization

• Based on PCR technology

• Based on restriction digest and PCR

• Based on DNA sequencing and microarray

Based on DNA-DNA hybridization

• RFLP, restriction fragment length polymorphism

• VNTR, variable number of tandem repeats

Based on PCR technology • Based on random primers

– RAPD, random amplified polymorphismic DNA

– AP-PCR, arbitrarily primed PCR

– DAF, DNA amplification fingerprinting

– ISSR, inter-simple sequence repeats

• Based on special primers – SSR, simple sequence repeats

– SCAR, sequence characterized amplified region

– STS, sequence-tagged site

– RGA, resistance gene analogs

The molecular basic of DNA marker

1. Point mutation between restriction sites (PCR primer binding sites)

2. Insertion between restriction sites (PCR primer binding sites)

3. Deletion between restriction sites (PCR primer binding sites)

4. Number of tandem repeats varying between restriction sites (PCR primer binding sites)

5. Single nucleotide mutation

restriction site

PCR primer

tandem repeats

Insertion

deletion

Restriction Fragment Length Polymorphism (RFLP)

•A Restriction Fragment Length Polymorphism , or RFLP, is a variation in the DNA sequence of a genome that can be detected by cutting the DNA into pieces with restriction enzymes and analyzing the size of the resulting fragments by gel electrophoresis.

•RFLPs are detected by fragmenting a sample of DNA using a restriction enzyme which can recognize and cut DNA wherever a specific short sequence occurs.

•The resulting DNA fragments are then separated by length though gel electrophoresis, and transferred to a membrane using the Southern Blot Hybridization method.

•Then the Length of the fragments is determined using complementarily labeled DNA probe.

•Fragment lengths vary depending on the location of the restriction sites.

•Each fragment length (band) can be used in the characterization of genetic diversity.

•RFLPs are generally to be moderately polymorphic.

•In addition to their high genomic abundance and their random distribution, RFLPs have the advantages of showing co-dominant alleles and having .

•The method has several disadvantages as well.

•The methodological procedures for RFLPs are expensive, laborious and require high skilled personal.

•In addition, if the research is conducted with poorly studied crops or wild species, suitable probes may not yet be available. •The procedures also requires large quantities of purified,

high molecular weight DNA for each digestion.

•Species with large genome will need more time to probe each blot .

•RFLPs are not amenable to automation, and collaboration among research teams requires distribution of the probes.

Restriction Fragment Length Polymorphism(RFLP)

1. mutation in restriction site

2. insertion mutation

3. Deletion mutation

restriction site

probe

Wild type

Mutant

Variable Numbers of Tandem Repeats(VNTR)

Restriction digest

Hybridization with tandem repeats sequence as probe

autoradiography

Restriction site

Core repeat sequences

Random Amplified Polymorphic DNA (RAPD)

•The method termed random amplification of polymorphic DNA (RAPD) uses a polymerase chain reaction (PCR) machine to produce many copies (amplification ) of random DNA segments called random amplified polymorphic DNA (also RAPD).

•Several arbitrary, short primers (8-12 nucleotides) are created and applied in the PCR using a large template of genomic DNA, hoping that fragments will amplify.

•By resolving the resulting patterns using agarose gel and ethidium bromide staining, a semi-unique profile can be gleaned from a RAPD reaction.

•Unlike traditional PCR analysis, RAPD does not require any specific knowledge of the DNA sequence of the target organism: the identical 10-mer primers will or will not amplify a segment of DNA, depending on positions that are complementary to the primers' sequence.

•For example, no fragment is produced if primers annealed too far apart or 3' ends of the primers are not facing each other.

• Therefore, if a mutation has occurred in the template DNA at the site that was previously complementary to the primer, a PCR product will not be produced, resulting in a different pattern of amplified DNA segments on the gel.

Limitations of RAPD

• Nearly all RAPD markers are dominant, i.e. it is not possible to distinguish whether a DNA segment is amplified from a locus that is heterozygous (1 copy) or homozygous (2 copies). Codominant RAPD markers, observed as different-sized DNA segments amplified from the same locus, are detected only rarely.

• PCR is an enzymatic reaction, therefore the quality and concentration of template DNA, concentrations of PCR components, and the PCR cycling conditions may greatly influence the outcome. Thus, the RAPD technique is notoriously laboratory dependent and needs carefully developed laboratory protocols to be reproducible.

• Mismatches between the primer and the template may result in the total absence of PCR product as well as in a merely decreased amount of the product. Thus, the RAPD results can be difficult to interpret.

Random Amplified Polymorphic DNA (RAPD)

1. Point mutation in PCR primer binding site -1

2. Point mutation in PCR primer binding site -2

3. Insertion mutation

4. Deletion mutation

primer Wild type

Mutant

Simple Sequence Repeats(SSRs)

•Simple Sequence Repeats(SSRs) or microsatellites, are polymorphic loci presented in nuclear and organellar DNA. They consist of repeating units of 1-6 base pair in length.

• They are multi-allelic and co-dominant.

•SSRs are used in population studies, genetic diversity analysis and to look for duplications or deletions of a particular genetic region.

•Microsatellites can be amplified through PCR, using the unique sequences of flanking regions as primers.

•Point mutation in the primer annealing sites in such species may lead to the occurrence of ‘null alleles’, where microsatellites fail to amplify in PCR assays.

Importance of characterization and evaluation

Information derived from characterization and evaluation of germplasm collection can be used to:

•Identify an accession

•Monitor identify of an accession over a number of regenerations

•Locate specific traits

•Assess genetic diversity of the collection

•Fingerprint genotypes

•Identify duplications

•Determine gap in the collection

•Facilitates preliminary selection of germplasm by end-users

•Study genetic diversity and taxonomic relationships

•Develop core collection

Simple Sequence Repeat (SSR)

Developing SSR Primers

Genome DNA

Digested fragments cloned to plasmid vector

Hybridized by poly GA/CT probe

Extract plasmid DNA from positive clones

Sequencing of cloned fragments

Designing primers according to flanking sequence

Based on restriction digest and PCR

• AFLP, amplified fragment length polymorphism

• CAPS, cleaved amplified polymorphic sequence

Amplified Fragments Length Polymorphism(AFLP)

•Amplified Fragments Length Polymorphism(AFLP) are DNA fragments obtained by using restriction enzymes to cut genomic DNA, followed by ligation of adaptors to the sticky ends of the restriction fragments.

•The amplified fragments are visualized an denaturing polyacrylamide gels either through autoradiography or via fluorescence methodologies.

•AFLP has many advantages compare with other marker technologies.

•AFLP-PCR is a highly sensitive method for detecting polymorphisms in DNA.

•AFLP has higher reproductively, resolution and sensitively at the whole genome level compared to other techniques.

•It also has the capacity to amplify between 50 and 100 fragments at one time. In addition, no prior sequence information is needed for amplification

•AFLP is widely used for the identification of genetic variation in strains or closely related plant species.

•The AFLP technology has been used in population genetics to determine slight differences within populations, as well as in linkage studies to generate maps for quantitative trait locus(QTL) analysis.

•AFLPs can be applied in studies involving genetic identity, parentage and identification of clones and cultivars, and phylogenetic studies of closely related species.

•The disadvantage of AFLP includes the need for purified, high molecular weight DNA, the dominance of alleles, and the possible non-homology of comigrating fragments belonging to different loci.

Procedure of AFLP

Pre-selective amplification

Selective amplification

Denatured Gel Electrophoresis

Based on DNA sequencing and microarray

• SNP, single nucleotide polymorphism

– SSCP (Single-strand conformation polymorphism)

– DGGE (Denaturing gradient gel electrophoresis)

– ASA (Allele-specific amplification)

– GBA (Genetic bit analysis)

– Oligonucleotide chip-based hybridization

– MALDI-TOF MS (Matrix assisted laser desorption ionization, time of flight mass spectrometry)

Population Development

Phenotypic Data

Marker Implementation

Donor Screening

Data Analysis

Association Analysis QTL Mapping

Genotypic Data

Marker Development for Molecular Breeding

Marker Identification

Molecular Breeding

SSR is repeating sequences of 2-5 (most of them) base pairs of DNA such as (AT)n, (CTC)n, (GAGT)n, (CTCGA)n Tool: SSRLocator, BatchPrimer3, MEGA6, BioEdit

SNP is a single nucleotide (A, T, C or G) mutation, and can be discovered from PCR, Next generation sequencing (NGS) such as RNA-Seq, RAD-Seq, GBS. Tool: BioEdit, DNASTAR, SAMtools, SOAPsnp, or GATK

Marker Discovery (SNP, SSR)

Genetic Diversity

Linkage/QTL Mapping

Genome-wide Selection

Marker-assisted Selection

Association Analysis

Genetic Map Construction

Molecular Plant Breeding Approach

Marker Identification (SNP, SSR Markers)

Molecular Breeding

Genetic diversity

Genetic Map

QTL mapping

Association analysis

SNP markers

MAS/GWS

SNP Add

effect

Dom

effect LOD

R^2

(%)

CoP930721_82 -0.123 -0.122 4.463 6.1

CoP930934_82 -0.076 0.274 2.807 3.9

Marker-assisted Selection

Marker-assisted Selection (MAS): using marker(s) to select trait of interest.

Marker type: SSR and SNP

QTL mapping : Linkage analysis

Marker Identification

Association Analysis Marker: trait

Marker Implementation

Parent selection and progeny testing

Marker-assisted backcrossing

Gene-pyramiding

Early generation selection for simple trait

Cultivar identity/assessment of ‘purity’

Late generation selection for complex trait

Fall 2016 HORT6033

10/31/2016

Marker Assisted Selection(MAS) In plant breeding

• Marker assisted selection refers to the manipulation of genomic regions that are involved in the expression of traits of interest through molecular markers.

• MAS of parental Lines for trait improvement:

• Molecular markers can be used to genotype a set of germplasm and the data used to estimate the genetic divergence among the evaluated materials

Use of Molecular Markers

Clonal identity,

Family structure,

Population structure,

Phylogeny (Genetic Diversity)

Mapping

Parental analysis,

Gene flow,

Hybridisation

Foreground selection and background selection using molecular marker

Molecular markers are now increasingly being employed to trace the presence of the target genes(foreground selection) as well as for accelerating the recovery of the recurrent parent genome(background selection) in backcross program.

MAS for improvement of qualitative traits:

MAS in developing quality protein maize(QPM) genotypes

MAS for improvement of quantitative traits

• MAS for improving heterotic performance in maize

• MAS for drought tolerance in maize

• Germplasm enhancement in tomatousing AB_QTL

• QTL mapping

• Single marker approach

• Simple interval mapping (SIM)

• Composite interval mapping (CIM)

• Application of biotechnology in plant breeding

• Somclonal variation

• Directed selection

• Haploidy

• Gene transfer

• Germplasm and pedigree identification

Jian-Long Xu, Institute of Crop Sciences, CAAS. Molecular Marker-assisted Breeding in Rice

Jian-Long Xu, Institute of Crop Sciences, CAAS. Molecular Marker-assisted Breeding in Rice

Population Size for MAS

Equation to Estimate Sample Size Required for QTL Detection

Useful when the gene(s) of interest is difficult

to select:

1. Recessive Genes

2. Multiple Genes for Disease Resistance

3. Quantitative traits

4. Large genotype x environment interaction

Marker Assisted Selection

MARKER ASSISTED

BREEDING SCHEMES

1. Marker-assisted backcrossing

2. Pyramiding

3. Early generation selection

4. ‘Combined’ approaches

Marker-assisted backcrossing

(MAB) • MAB has several advantages over conventional

backcrossing:

– Effective selection of target loci

– Minimize linkage drag

– Accelerated recovery of recurrent parent

1

2

3

4

Target locus

1

2

3

4

RECOMBINANT SELECTION

1

2

3

4

BACKGROUND SELECTION

TARGET LOCUS SELECTION

FOREGROUND SELECTION

BACKGROUND SELECTION

Gene Pyramiding

Widely used for combining multiple disease resistance genes for specific races of a pathogen

Pyramiding is extremely difficult to achieve using conventional methods Consider: phenotyping a single plant for multiple forms of seedling resistance – almost impossible

Important to develop ‘durable’ disease resistance against different races

F2

F1

Gene A + B

P1

Gene A

x P1

Gene B

MAS

Select F2 plants that have

Gene A and Gene B

Genotypes

P1: AAbb P2: aaBB

F1: AaBb

F2 AB Ab aB ab

AB AABB AABb AaBB AaBb

Ab AABb AAbb AaBb Aabb

aB AaBB AaBb aaBB aaBb

ab AaBb Aabb aaBb aabb

Process of combining several genes, usually from 2 different parents,

together into a single genotype

x

Breeding plan

Early generation MAS

• MAS conducted at F2 or F3 stage

• Plants with desirable genes/QTLs are selected and

alleles can be ‘fixed’ in the homozygous state

– plants with undesirable gene combinations can be

discarded

• Advantage for later stages of breeding program

because resources can be used to focus on fewer

lines

F2

P2

F1

P1 x

large populations (e.g. 2000 plants)

Resistant Susceptible

MAS for 1 QTL – 75% elimination of (3/4) unwanted genotypes

MAS for 2 QTLs – 94% elimination of (15/16) unwanted genotypes

P1 x P2

F1

PEDIGREE METHOD

F2

F3

F4

F5

F6

F7

F8 – F12

Phenotypic screening

Plants space-planted in rows for individual plant selection

Families grown in progeny rows for selection.

Preliminary yield trials. Select single plants.

Further yield trials

Multi-location testing, licensing, seed increase and cultivar release

P1 x P2

F1

F2

F3

MAS

SINGLE-LARGE SCALE MARKER-

ASSISTED SELECTION (SLS-MAS)

F4 Families grown in progeny rows for selection.

Pedigree selection based on local needs

F6

F7

F5

F8 – F12 Multi-location testing, licensing, seed increase and cultivar release

Only desirable F3 lines planted in field

Benefits: breeding program can be efficiently

scaled down to focus on fewer lines

Combined approaches

• In some cases, a combination of phenotypic screening and MAS approach may be useful

1. To maximize genetic gain (when some QTLs have been unidentified from QTL mapping)

2. Level of recombination between marker and QTL (in other words marker is not 100% accurate)

3. To reduce population sizes for traits where marker genotyping is cheaper or easier than phenotypic screening

‘Marker-directed’ phenotyping

BC1F1 phenotypes: R and S

P1 (S) x P2 (R)

F1 (R) x P1 (S)

Recurrent

Parent

Donor

Parent

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 …

SAVE TIME & REDUCE

COSTS

*Especially for quality traits*

MARKER-ASSISTED SELECTION (MAS)

PHENOTYPIC SELECTION

(Also called ‘tandem selection’)

Use when markers are not

100% accurate or when

phenotypic screening is

more expensive compared

to marker genotyping

Crop characterization by identifying isozymes

• Enzymes electrophoresis relies on quantifying a series of enzymes that are present in a specific tissue such as germinating seedlings

• Within each enzyme measured different alleles(allozymes or isozymes) can be measured by their differential migration on a starch or polyacrylamide gel

• Enzyme electrophoresis refers to the migration of proteins(enzymes) from a starting point at the base of the gel and across an electric field.

• The amount of migration is dependent on the molecular weight of the enzyme, charge differences, and three-dimensional structure.

• Early studies in maize could resolve approximately 85% of a sample of inbred with known pedigrees (Stuber and Goodman, 1983).Smith et al.(1987)were able to distinguish 94% of 62 inbred lines of known pedigree.

• Furthermore, these inbred could be identified in hybrid combinations and hybrid yield could be predicted based on their enzymes profile.

• Biochemical data is generally accepted as one method of identifying germplasm and in at least one legal case in United States has been used to verify ownership of a maize inbred .

Advanced tools for Plant Breeding

• Mutagenesis

• Tissue culture

• Haploidy

• In situ hybridization

• DNA markers

Advanced technology

• Molecular markers

• Marker-assisted selection

• DNA sequencing

• Plant genomic analysis

• Bioinformatics

• Microarray analysis

• Primer design

• Plant transformation

Modern Breeding Tools

Increase of breeding effectiveness and efficiency

In vitro culture Genomic tools Genomic engineering

Future Challenges

Challenge: Increase of human population by 60-80%, requiring to

nearly double the global food production

Multidisciplinary Field

Biometry/ Statistics

Pathology