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
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