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

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)

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

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

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

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.

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

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

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

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

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

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

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?

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-

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

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)

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

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