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 Concept Plant breeding is the process by which humans change certain aspects of plants over time in order to introduce desired characteristics 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 Mendel Empirical evidence on heredity Watson, Crick, Wilkins & Rosalind Franklin model for DNA structure 1694 1866 1923 1953 Camerarius crossing as a method to obtain new plant types Wallace First commercial hybrid corn
The Green Revolution (1960) Challenge: improve wheat and maize to meet the production needs of developing countries Norman Borlaug 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 Abiotic and biotic resistance breeding (disease/pest resistance, drought and salt tolerance) P 1 x BC 1 F 1 Backcross breeding Classic Breeding Main Street F 2 F 3 F 4-5 F 6-7 P 1 x P 2 F 1 F 8-10 Preliminary Cultivar variety Release Molecular breeding Parent selection Predictive breeding True/false, self testing MAS for simple traits MAS for quantitative traits Final Yield Test 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 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 T 4 1.6 10 2 800 0.2 E. coli 4.2 10 3 1,750 2.4 Yeast 2.0 10 4 4,200 4.8 Fungus 2.7 10 4 1,000 27.0 Nematode 8.0 10 4 320 250.0 Drosophila 1.4 10 5 280 500.0 Rice 4.5 10 5 1,500 300.0 Mouse 3.0 10 6 1,700 1,800.0 Human race 3.3 10 6 3,300 1,000.0 Maize 2.5 10 6 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 10 6 3300 1000 4.5 10 5 1500 300 2.5 10 6 2500 1000 7.0 10 4 500 140 7.1 10 5 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) Insertion 3. Deletion between restriction sites (PCR primer binding sites) deletion 4. Number of tandem repeats varying between restriction sites (PCR primer binding sites) 5. Single nucleotide mutation restriction site PCR primer tandem repeats
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)
Marker Development for Molecular Breeding Donor Screening Population Development Phenotypic Data Genotypic Data Data Analysis QTL Mapping Association Analysis Marker Identification Marker Implementation Molecular Breeding
Molecular Plant Breeding Approach 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) 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 Genetic diversity Genetic Diversity Genetic Map Construction QTL mapping Association analysis MAS/GWS Association Analysis Marker Identification (SNP, SSR Markers) Linkage/QTL Mapping Genetic Map SNP Add effect Dom effect LOD R^2 (%) Marker-assisted Selection Genome-wide Selection CoP930721_82-0.123-0.122 4.463 6.1 CoP930934_82-0.076 0.274 2.807 3.9 Molecular Breeding SNP markers
Fall 2016 HORT6033 10/31/2016 Marker-assisted Selection Marker-assisted Selection (MAS): using marker(s) to select trait of interest. Marker type: SSR and SNP QTL mapping : Linkage analysis Association Analysis Marker: trait Marker Identification Marker Implementation Parent selection and progeny testing Marker-assisted backcrossing Early generation selection for simple trait Late generation selection for complex trait Gene-pyramiding Cultivar identity/assessment of purity
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 Jian-Long Xu, Institute of Crop Sciences, CAAS. Molecular Marker-assisted Breeding in Rice Equation to Estimate Sample Size Required for QTL Detection
Marker Assisted Selection 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 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 1 2 3 4 1 2 3 4 Target locus TARGET LOCUS SELECTION RECOMBINANT SELECTION BACKGROUND 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
Process of combining several genes, usually from 2 different parents, together into a single genotype Breeding plan Genotypes P 1 Gene A x P 1 Gene B P 1 : AAbb x P 2 : aabb F 1 Gene A + B F 1 : AaBb F 2 MAS Select F2 plants that have Gene A and Gene B F 2 AB Ab ab ab AB AABB AABb AaBB AaBb Ab AABb AAbb AaBb Aabb ab AaBB AaBb aabb aabb ab AaBb Aabb aabb aabb
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
Susceptible P 1 x P 2 Resistant F 1 F 2 large populations (e.g. 2000 plants) MAS for 1 QTL 75% elimination of (3/4) unwanted genotypes MAS for 2 QTLs 94% elimination of (15/16) unwanted genotypes
PEDIGREE METHOD P1 x P2 F1 SINGLE-LARGE SCALE MARKER- ASSISTED SELECTION (SLS-MAS) P1 x P2 F1 F2 Phenotypic screening F2 MAS F3 Plants spaceplanted in rows for individual plant selection F3 Only desirable F3 lines planted in field F4 Families grown in progeny rows for selection. F4 Families grown in progeny rows for selection. F5 F5 Pedigree selection based on local needs F6 Preliminary yield trials. Select single plants. F6 F7 Further yield trials F7 F8 F12 Multi-location testing, licensing, seed increase and cultivar release F8 F12 Multi-location testing, licensing, seed increase and cultivar release 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 (Also called tandem selection ) Recurrent Parent P 1 (S) x P 2 (R) F 1 (R) x P 1 (S) Donor Parent BC 1 F 1 phenotypes: R and S MARKER-ASSISTED SELECTION (MAS) Use when markers are not 100% accurate or when phenotypic screening is more expensive compared to marker genotyping 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 PHENOTYPIC SELECTION SAVE TIME & REDUCE COSTS *Especially for quality traits*
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 In vitro culture Genomic tools Genomic engineering Increase of breeding effectiveness and efficiency
Future Challenges Multidisciplinary Field Pathology Challenge: Increase of human population by 60-80%, requiring to nearly double the global food production Biometry/ Statistics