Plant breeding Methods and use of classical plant breeding. … · 2018-03-15 · Plant breeding...
Transcript of Plant breeding Methods and use of classical plant breeding. … · 2018-03-15 · Plant breeding...
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
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.
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
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