THE USE OF GENETIC MARKERS IN PLANT BREEDING

Use of Molecular Markers Clonal identity, Family structure, Population structure, Phylogeny (Genetic Diversity) Mapping Parental analysis, Gene flow, Hybridisation

Genetic Diversity Define appropriate geographical scales for monitoring and management (epidemology) Establish gene flow mechanism Identify the origin of individual (mutation detection) Monitor the effect of management practices Manage small number of individual in ex situ collection Establish of identity in cultivar and clones (fingerprint) Paternity analysis and forensic

Genetic Diversity

Clonal Identity early selection of the good allele fingerprints seeds, plantlets

A linear order of genes or DNA fragments Mapping The determination of the position and relative distances of gene on chromosome by means of their linkage Genetic map A linear arrangement of genes or genetic markers obtained based on recombination Physical map A linear order of genes or DNA fragments

It contains ordered overlapping cloned DNA fragment Physical Mapping It contains ordered overlapping cloned DNA fragment The cloned DNA fragments are usually obtained using restriction enzyme digestion

Chromosomes with morphological Chromosomes with molecular Genetic Maps Molecular markers (especially RFLPs and SSRs) can be used to produce genetic maps because they represent an almost unlimited number of alleles that can be followed in progeny of crosses. R r T t or Chromosomes with morphological marker alleles RFLP1a RFLP2a RFLP4a RFLP3a SSR1a SSR2a RFLP1b RFLP2b RFLP4b RFLP3b SSR1b SSR2b Chromosomes with molecular marker alleles

QTL (Quantitative Trait Loci) A locus or DNA segment that carries more genes coding for an agronomic or other traits Individual loci responsible for quantitative genetic variation Region in the genome containing factors influencing a quantitative trait Region identified by statistical association QTL Mapping A set of procedures for detecting genes controlling quantitative traits (QTL) and estimating their genetics effects and location Localizing and determining a segment of DNA that regulate quantitative traits Detecting and locating gene having an effect on a quantitative traits To assist selection Marker Assisted Selection

Multigenic trait; ex: plant growth =Quantitative Trait Loci Types of traits Single gene trait: seed shape Multigenic trait; ex: plant growth =Quantitative Trait Loci

Linkage groups

Developing a Marker Best marker is DNA sequence responsible for phenotype i.e. gene If you know the gene responsible and has been isolated, compare sequence of wild-type and mutant DNA Develop specific primers to gene that will distinguish the two forms

Developing a Marker If gene is unknown, screen contrasting populations Use populations rather than individuals Need to “blend” genetic differences between individual other than trait of interest

Developing Markers Cross individual differing in trait you wish to develop a marker Collect progeny and self or polycross the progeny Collect and select the F2 generation for the trait you are interested in Select 5 - 10 individuals in the F2 showing each trait

→ Near Isogenic Lines, Recombinant Inbreeds, Single Seed Decent Developing Markers Extract DNA from selected F2s Pool equal amounts of DNA from each individual into two samples - one for each trait Screen pooled or “bulked” DNA with what method of marker method you wish to use Conduct linkage analysis to develop QTL Marker Other methods to develop population for markers exist but are more expensive and slower to develop → Near Isogenic Lines, Recombinant Inbreeds, Single Seed Decent

DNA markers can reliably predict phenotype MAS Marker assisted selection The use of DNA markers that are tightly-linked to target loci as a substitute for or to assist phenotypic screening Assumption DNA markers can reliably predict phenotype

Marker Assisted Selection Breeding for specific traits in plants is expensive and time consuming The progeny often need to reach maturity before a determination of the success of the cross can be made The greater the complexity of the trait, the more time and effort needed to achieve a desirable result The goal to MAS is to reduce the time needed to determine if the progeny have trait The second goal is to reduce costs associated with screening for traits If you can detect the distinguishing trait at the DNA level you can identify positive selection very early.

CONVENTIONAL PLANT BREEDING x Donor Recipient F1 large populations consisting of thousands of plants F2 PHENOTYPIC SELECTION Salinity screening in phytotron Bacterial blight screening Phosphorus deficiency plot Field trials Glasshouse trials

MARKER-ASSISTED BREEDING P1 x P2 Susceptible Resistant F1 large populations consisting of thousands of plants F2 MARKER-ASSISTED SELECTION (MAS) Method whereby phenotypic selection is based on DNA markers

Advantages of MAS Simpler method compared to phenotypic screening Especially for traits with laborious screening May save time and resources Selection at seedling stage Important for traits such as grain quality Can select before transplanting in rice Increased reliability No environmental effects Can discriminate between homozygotes and heterozygotes and select single plants

Potential benefits from MAS more accurate and efficient selection of specific genotypes May lead to accelerated variety development more efficient use of resources Especially field trials Crossing house Backcross nursery

Overview of ‘marker genotyping’ (1) LEAF TISSUE SAMPLING (2) DNA EXTRACTION (3) PCR (4) GEL ELECTROPHORESIS (5) MARKER ANALYSIS

Developing a Marker Best marker is DNA sequence responsible for phenotype i.e. gene If you know the gene responsible and has been isolated, compare sequence of wild-type and mutant DNA Develop specific primers to gene that will distinguish the two forms

Developing a Marker If gene is unknown, screen contrasting populations Use populations rather than individuals Need to “blend” genetic differences between individual other than trait of interest

Developing Markers Cross individual differing in trait you wish to develop a marker Collect progeny and self or polycross the progeny Collect and select the F2 generation for the trait you are interested in Select 5 - 10 individuals in the F2 showing each trait

→ Near Isogenic Lines, Recombinant Inbreeds, Single Seed Decent Developing Markers Extract DNA from selected F2s Pool equal amounts of DNA from each individual into two samples - one for each trait Screen pooled or “bulked” DNA with what method of marker method you wish to use Conduct linkage analysis to develop QTL Marker Other methods to develop population for markers exist but are more expensive and slower to develop → Near Isogenic Lines, Recombinant Inbreeds, Single Seed Decent

Considerations for using DNA markers in plant breeding Technical methodology simple or complicated? Reliability Degree of polymorphism DNA quality and quantity required Cost** Available resources Equipment, technical expertise

Markers must be tightly-linked to target loci! Ideally markers should be <5 cM from a gene or QTL Marker A QTL 5 cM RELIABILITY FOR SELECTION Using marker A only: 1 – rA = ~95% Marker A QTL Marker B 5 cM Using markers A and B: 1 - 2 rArB = ~99.5% Using a pair of flanking markers can greatly improve reliability but increases time and cost

Markers must be polymorphic RM84 RM296 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 P1 P2 P1 P2 Not polymorphic Polymorphic!

DNA extractions LEAF SAMPLING Mortar and pestles Porcelain grinding plates LEAF SAMPLING Wheat seedling tissue sampling in Southern Queensland, Australia. High throughput DNA extractions “Geno-Grinder”

Agarose or Acrylamide gels PCR-based DNA markers Generated by using Polymerase Chain Reaction Preferred markers due to technical simplicity and cost PCR Buffer + MgCl2 + dNTPS + Taq + Primers + DNA template PCR THERMAL CYCLING GEL ELECTROPHORESIS Agarose or Acrylamide gels

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 Marker-assisted backcrossing Pyramiding Early generation selection ‘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 RECOMBINANT SELECTION 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

Select F2 plants that have Gene A and Gene B Process of combining several genes, usually from 2 different parents, together into a single genotype Breeding plan Genotypes P1 Gene A x P1 Gene B P1: AAbb x P2: aaBB F1 Gene A + B F1: AaBb F2 F2 AB Ab aB ab AABB AABb AaBB AaBb AAbb Aabb aaBB aaBb aabb MAS Select F2 plants that have Gene A and Gene B

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

large populations (e.g. 2000 plants) x P2 Susceptible Resistant F1 F2 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

SINGLE-LARGE SCALE MARKER-ASSISTED SELECTION (SLS-MAS) PEDIGREE METHOD 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 P1 x P2 F1 Phenotypic screening F2 Plants space-planted in rows for individual plant selection F3 Families grown in progeny rows for selection. F4 F5 Preliminary yield trials. Select single plants. F6 Further yield trials F7 Multi-location testing, licensing, seed increase and cultivar release F8 – F12 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 To maximize genetic gain (when some QTLs have been unidentified from QTL mapping) Level of recombination between marker and QTL (in other words marker is not 100% accurate) 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 P1 (S) x P2 (R) Donor Parent Use when markers are not 100% accurate or when phenotypic screening is more expensive compared to marker genotyping F1 (R) x P1 (S) BC1F1 phenotypes: R and S MARKER-ASSISTED SELECTION (MAS) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 … SAVE TIME & REDUCE COSTS PHENOTYPIC SELECTION *Especially for quality traits*