Introduction to molecular markers for breeding of fruit tree speciesAndrea Patocchi (Agroscope)

Content

• What is a molecular marker• Three examples of molecular markers• Examples of applications and limits of

molecular markers• Summary

What is a molecular markers?

Molecular markers associated to a gene are naturally occurring DNA sequences that are close to the specific genes

In general, the DNA sequence of the gene of interest it is not known, while the sequence of the marker associated to it is known

chromosome DNA

cell

marker for gene 1

gene 1

Adapted from: http://biointeraction.blogspot.ch/2010/09/dna-and-chromosome.html

How to find the association …

… between a trait and molecular markers?

Necessary are:- A good coverage of the genome with molecular markers- A precise phenotyping of the progeny

An insufficient coverage with markers leads to weak association

Mistakes during phenotyping lead to wrong associations (map positions)

Adapted from Collard et al. 2005

Phenotyping e.g. inoculation with scab of the progeny plants

scoring and codingABBAABBBABBBAAAAABB

R-gene

Note: identical score as marker G

Use in breeding

Most of the times molecular markers are used to make predictions:Is a specific marker (allele) present, with a determinate probability, is also the gene (allele) of interest present

The closer (the more associated) are the molecular marker and the gene of interest, the higher will be the probability of a correct prediction

The “perfect” marker is a marker developed within the sequence of the gene

The distance of a marker and the gene of interest (or between two markers) is expressed in centimorgan (cM):

1cM = 1 wrong prediction in 100 cases

The most used application of markers in breeding is theMarker Assisted Selection (MAS)

Marker 1 for gene 1

Gene 1

Marker 2 for gene 1

«perfect» marker for gene 1

Three types of molecular markers

Using methods of the molecular biology (polymerase chain reaction; PCR) a DNA fragment is multiplied, and made visible

The most used molecular markers in marker assisted selection are:• Sequence Characterized Amplified Regions (SCARs)• Simple Sequence Repeats (SSRs, or microsatellites)• Single Nucleotide Polymorphism (SNP)

Characteristics of the three types of markers

SCAR: have in general only two alleles; alleles show presence/absence polymorphism or differs greatly by size

SSR: have often > 10 alleles; the alleles show differences of the length of the repeated sequence (e.g. CTT);

allele 1 …ATGCTTATCGG[CTTCTTCTTCTTCTTCTTCTT]GATCAAATTACCCGTAGATA… CTT X7allele 2 …ATGCTTATCGG[CTTCTTCTTCTTCTTCTTCTTCTT]GATCACATTACCCGTAGATA… CTT X8allele 3 …ATGCTTATCGG[CTTCTTCTTCTTCTTCTTCTTCTTCTTCTT]GATCACATTACCCGTAGATA… CTT X10

SNP: have in general only two alleles; their sequence differ only by a single nucleotide (null allele also possible)

allele 1 …ATGCTTATCGGGATCAAATTACCCGTAGATA… allele 2 …ATGCTTATCGGGATCACATTACCCGTAGATA…

Present/absence of a specific band,only one allele is amplified, dominant marker

Co-dominant SCAR marker: it allows to distinguish between homo- and heterozygous plants

Examples of applications (1)

Verification of pedigrees• Allele 159bp of SSR marker

CH-Vf1 is associated to Vf• From Florina to F2 26829-2-2

the pedigree is ok BUT• F2 26829-2-2 looks not to be a

product of a sib cross (allele 137bp (*) is not present in Mf821 or Rome Beauty)

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ases

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Rom

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PRI 1

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I 612

-1 (V

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Vf allele

outbreeder

Adapted from Vinatzer et al. 2004

Examples of applications (2)

…in a cross between two genotypes heterozygous for Vf• Allele 159bp of SSR marker

CH-Vf1 is associated to Vf• Three progeny plants are

outbreeders (probably from the same father)

Identification of homozygous genotypes…

outbreedersAllele 159bpAssociated to Vf scab resistance

Adapted from Vinatzer et al. 2004

Examples of applications (3)

…in a cross between two genotypes heterozygous for Rvi2 and Rvi6, respectively

Without molecular markers this work can only be done if virulent isolates to both R-genes are available, BUT is extremely time consuming!

Identification of pyramids of two R-genes (Rvi2&6)

Rvi2 rvi2

Rvi6 Rvi2/Rvi6 rvi2/Rvi6

rvi6 Rvi2/rvi6 rvi2/rvi6

M P1 P2 S1 S2 S3 S4 S5

Incr

easi

ng s

ize o

f the

ban

ds

Rvi2 marker

Rvi6 marker

Examples of applications (4)

e.g. peach:• peach/necatrine phenotype;• yellow/white flesh• Flat/round fruit shape

Early selection for traits that cannot be assessed at seedling stage (fruit traits)

Examples of applications (5) / Limits

… with a marker having an allele highly specific for the allele of the gene of interest (e.g. Rvi6/Vf resistance, SSR CH-Vf1)Caution!The presence of the R-gene (allele) in the genotypes amplifying the allele associated to the R-gene (allele) NEEDS to be validated:• Are the plants really resistant and showing the

typical symptoms? • Is it plausible from the pedigree that the

genotype is carrying the R-gene?

Screening of collections

Adapted from Vinatzer et al. 2004

Summary

• Molecular markers are very useful tools for breeding• To get efficient and good molecular markers for MAS, we need

good phenotyping and good and many markers (2 outputs from FruitBreedomics)

• Molecular markers allows to make predictions that cannot be done without them (e.g. pyramids or R-genes,…)

• They allows to save money by an early identification of progeny plants having a desired combination of traits

Thank you for your attention