1. Chapter 7: Molecular markers in breeding
Why use molecular markers in breeding?
For which type of applications? Where?
Which type of molecular markers? How?

2. Chapter 7: Molecular markers in breeding
With many thanks to Isabel Roldán-Ruiz 1 for allowing me to use information and pictures from the slides of her course ()
1 Institute for Agricultural and Fisheries Research ILVO (Research institute of Ministry of the Flemish Community)

3. Why?  The phenotype is an imperfect predictor of the genotype:
possibly unobservable before the time selection must be made
the phenotype of a plant is always the expression of its genotype, but with a relatively big influence of the environment
The phenotype is not effective in resolving negative associations between genes (linkage, epistasis)
Genotype
Environment
Breeder’s black box:
Number of genetic factors
Relative importance of each factor for the determination of the phenotype
Interactions among factors (pleiotropism, epistasis, linkage, association)
The breeder works with ‘phenotypes’, and makes inferences about ‘genotypes’
Phenotype

4. DNA markers = direct reflection of genotype
Why?
DNA markers = direct reflection of genotype 
Two plants can display similar phenotypes, but be very different from a genetic point of view
e.g. Lolium perenne genotypes with similar yield. In each genotype different genomic regions can be responsible for the high yield potential
Genotype A
Genotype B
same phenotype

5. DNA markers = direct reflection of genotype
Why?
DNA markers = direct reflection of genotype 
Two plants can display different phenotypes, but be very similar from a genetic point of view
e.g. Mutant genotypes in edible apple (Malus domestica). Columnar phenotype in cv. Telamon (carrier of a spontaneous mutation original found in cv. McIntosh) is caused by mutation at one single locus
Wild type
Mutant
Short inter-nodes
Reduced plant height
Different branching ….
Different phenotype

6. Why?
A molecular marker is a DNA sequence which can be readily detected and whose inheritance can be monitored.
For example in the case of pathogen resistance, a good marker avoids elaborate infection tests for the selection of resistant plants.
DNA-markers allow the breeder to introduce into their cultivated plant only the gene(s) of interest from a related species. While conventional breeding methods rely on the transfer of the whole genome (along the gene of interest, undesirable characters are also co-inherited and have to be eliminated through back crossing followed by selection) DNA-markers allow to eliminate in a few generations ‘undesired’ genome regions.

7. Why? 
The introgression of recessive alleles through classical back cross breeding is even more lengthy as this requires additional generations of selfing (to identify the homozygous recessive) after every back-crossing
Some characters like complex disease resistance reaction or biotic stresses (QTLs) that show continuous variation and do not fit into Mendelian ratios are most difficult to detect and transfer through conventional plant breeding, due to interactions with the environment

8. How can molecular markers help?
Why? 
How can molecular markers help?
Molecular markers allow working with genotype information directly
Analyze the effect of the genotype on the phenotype
Provide the breeder a tool to look into the ‘black box’ of the genotype

9. Genes and genomes Human Yeast Z. mays E. coli
Eukaryotic genomes contain much DNA outside the genes
Human
Yeast
Z. mays
E. coli
Genomes 2
T.A. Brown, Bios

10. Two basic types of DNA-markers
Which?
Two basic types of DNA-markers 
Causal mutations: the mutation is responsible for the change in the color of the flower
Mutation = Marker
- The most useful
- Difficult to find
Difficult to prove
- Less often used
Presumed non-functional DNA-markers, in the gene (but not the causal mutations) or linked to the gene
In some cases enough
Easier to find
Easier to prove
- Frequently used
Mutation
Marker M m

11. Where? Fields of application Cultivar identification Specific markers
Cultivar identification
Specific markers
Understanding relationships
Analysis of diversity
 Chapter 8
Construction of genetic linkage maps and tagging economically important genes
Marker assisted selection
e.g. Introgression, QTL-analysis
 Chapter 9, applications based on genetic maps

12. Where? Cultivar identification (fingerprint)
Cultivar identification (fingerprint)
Useful to control the identity of reproductive material (seeds, grafts, bulbs…)
Useful to control the non-authorized use of cultivars from other breeders

13. Where? Understanding germplasm relationships 
Markers are useful in four types of measurements needed in this field:
Identity: correct label of plants?
Similarity: degree of relatedness among plants?
Structure: is possible to identify groups of related plants?
Detection: posses some plants of the collection a particular allele of a gene?

14. Where? 
Construction of genetic linkage maps and tagging economically important genes
Segregating population constructed using plants with contrasting characteristics
Through linkage analysis (analysis of the co-segregation of marker alleles) a genetic map is constructed
By combining phenotypic data with genetic-map information, identify markers which are linked to the gene(s) responsible of the trait m- m- m- m- m- m- m- m- m-
Position of gene(s) responsible for the trait m- m- m- X m- m- m- m- m- m- m- m- m- m- m-

15. Where? 
Marker Assisted Selection and introgression through back-crossing
Use markers linked to agronomic traits to select the best plants to cross
In most cases, selection has to be performed for more than one trait simultaneously
In some cases we are interested in the introgression of a specific genome region

16. Where?  Quantitative trait
Multiple genes affect the expression of the trait
The expression in the population is a ‘bell-shaped’ curve: there are many genotypes and there are no clear phenotypic differences among them:
Height
Frequency distribution
If we want to find DNA-markers that can help us to predict this phenotype, we are searching for several genetic loci simultaneously
There exist standard experimental approaches for this (e.g. QTL analysis)

17. DNA markers: desired properties
How? 
DNA markers: desired properties
Highly polymorphic: able to detect many different alleles
Highly informative; if one individual carries two different alleles we can visualize both (co-dominant)
Occurrence throughout the studied genome, at high densities but not clustered
Easy, fast and inexpensive to screen
Reproducible within and between laboratories
 No single technique fulfills all these criteria
Choice of DNA analysis technique depends upon the infrastructure, technical expertise and operational funds available as well as requirements of the experiment

18. How? Identification individual: multilocus STR (of AFLP)
Analysis relationship:
Sequence if different species
AFLP within genus or species
Multilocus STR within species
Specific marker for diagnosis or marker assisted breeding PCR-RFLP or SCAR or SNP or STR
Overview whole genome (breeding, diversity): AFLP, multilocus STR or SNP