Welcome to the Training Seminar on:MAB in apple and peach, and FruitBreedomics Breeding Interface

Comacchio, 19th June 2015

Program of the day

Time Subject  Speaker(s)08:30 Short introduction on molecular markers for breeders A. Patocchi (Agroscope)

08:45 Sampling procedures. Hands on experience and practical details

I.Baumgartner (Agroscope)

09:00 Utilization of molecular markers for parental selection: Example on ASF-IRTA breeding program

P. Arus (IRTA)

09:30 Utilization for hybrids selection. Examples on apple   Pyramiding scab and mildew resistance in the Agroscope

breeding programI.Baumgartner (Agroscope)

  Selecting seedlings with good texture in the FEM breeding program

F. Costa (FEM)

10:30 Utilization for hybrids selection. Examples on peach   Selecting for acidity and flat shape M.J. Aranzana (IRTA)  Selecting for diseases and insects resistances T. Pascal (INRA)11:30 Coffee break 12:00 For what traits do we have markers available? A. Patocchi (Agroscope) /

M.J. Aranzana (IRTA)12:15 Comparing costs I. Batlle (IRTA)12:30 Lunch 13:30 FruitBreedomics MAB Services   Introduction F. Laurens (INRA)  Vegepolys F. Laurens (INRA)  FEM Platform F. Costa (FEM)  IRTA P. Arus (IRTA)14:00 FruitBreedomics Breeding Interface N. Nazzicari (PTP)15:00 Return to Bologna 16:00 Arrival to Bologna

Short introduction to molecular markers for breedingAndrea Patocchi (Agroscope)

Content

• What is a molecular marker?• Three examples of molecular markers• Few examples of application of molecular

markers• Take-home messages

What is a molecular marker?

Molecular markers associated to a gene are naturally occurring DNA fragments that are close to 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

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

chromosome DNA

cell

marker for gene 1

gene 1

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

In breeding molecular markers are used to make predictions

Kind of predictions:Is a specific marker (allele) present, with a determinate probability, is also the gene (allele) of interest present

The closer (the more associated) the molecular marker and the gene of interest are, 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

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 SCAR, SSR & SNP markers

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

SSR: co-dominant, 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: co-dominant, 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)

10 b

ases

ladd

er

Rom

e Be

auty

M. fl

orib

unda

821

(Vf

)F2

268

29-2

-2 (V

f)Go

lden

Del

icio

usPR

I 14-

126

(Vf)

Star

king

PRI 6

12-1

(Vf)

John

atan

Flor

ina

(Vf)

10 b

ases

ladd

er

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

easin

g siz

e of

the

band

s

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)

Take-home messages

• Molecular markers are very useful tools for breeding• Molecular markers allows to make predictions that cannot be

done without them (e.g. pyramids of 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