1. Real world application of markers
in peach breeding programs:
Marker Assisted Selection pilot studies on
green peach aphid Resistance (Rm2 gene)
Mauroux JB, Dievart V, Tuero C, Pascal T
YOUR LOGO

2. FB selected traits for whom MAB implémentation
is ongoing
Genetic linkage map of peach (8 chromosomes)
YOUR LOGO

3. Real world application of markers in peach breeding programs:
Pilot studies on Rm2
Marker assisted selection (MAS) refers to the use of DNA markers that are
tightly-linked to target loci as a substitute for or to assist phenotypic
screening.
Assumption: DNA markers can reliably predict phenotype.
YOUR LOGO

4. Implementation of MAS for the resistance of green aphid :
from gene mapping to marker validation
Marker
• From the resulting map, identification of a set of markers close to the Rm2 gene
Identification
Marker
Checking
Genetic
Test on
Offsprings
Marker
Validation
• Leaf sampling
• Genotyping parents with the 10 previously identified markers
• Screening of the set of markers on the parents of 6 targeted crosses
• Leaf sampling
• Genotyping individuals with the 2 selected markers
• Establishment of a list of resistant and susceptible individuals
• Reliability evaluation of the selected markers, by comparison between real
phenotypes and phenotypes predicted by markers
YOUR LOGO
MAB
Marker development
Gene
Mapping
• Development of a population (F2) segregating for resistance to the aphid
• Phenotypic testing (R + S) + genetic test
• Creation of a genetic map (markers + Rm2)

5. Development of a population (F2) segregating for
resistance to aphids
Genomic data
+
Phenotypic data
Genetic map
YOUR LOGO

6. Methodology for testing resistance to green peach aphid
Without markers
Aphids transfert
1 week
Mass rearing
(on 3 month seedlings)
Multiplication
(on GF305)
Aphid production
YOUR LOGO
Controlled infestation
+Phenotyping
Resistance test

7. Methodology for testing resistance to green peach aphid
Using markers
Aphids transfert
Susceptibles
Markers
1 week
Mass rearing
(on 3 month seedlings)
Multiplication
(on GF305)
Aphid production
Controlled infestation
+Phenotyping
Resistants
Resistance test
Advantages of markers:
 The production of aphids is not required (time/cost savings)
 possibility to know whether the resistant individuals have one or more resistance factors.
YOUR LOGO

8. Creation of a genetic map (markers + Rm2)
CH1
CH2
CH3
CH4
CH5
CH6
CH7
Rm2
Genetic map derived from the (Pamir x Rubira)2 cross
YOUR LOGO
CH8

9. From the resulting map, identification of a set of markers
close to the Rm2 gene
End of the chromosome 1

Rm2
Chromosome 1
Zoom +
mkr01
mkr02
mkr03
mkr04
mkr05
mkr06
mkr07
mkr08
mkr09
mkr10
Rm2 gene has been located close to
the end of the chromosome 1

Many markers identified in this area

10 markers were selected as good
candidates for MAB
YOUR LOGO

10. Screening of the set of markers on the parents of 6
targeted crosses
Parent of the pilot studies:
Rm2
Leaf sampling + Genetic Tests
or
Polymorphic marker
Monomorphic marker
YOUR LOGO

11. Leaf sampling

Samples of young leaves were collected in greenhouse at INRA of Avignon

A leaf punch collection device were used to normalize quantity of the collected material (8
leaf discs/tree)

Samples were placed in their appropriate positions in a 96-well plate

Plates were kept cool during all the collection process

Once every plants were collected, plates were packaged with silica gel (for desiccation) and
shipped to LGC genomics (for DNA extraction and genotyping)

Note : for sampling in orchard, we recommend to collect the leaves in plastic bags
- DNA extraction
- Genotyping
YOUR LOGO

12. Rm2
Rm2
Rm2
Establishment of a list of resistant and susceptible
individuals
Exemple of genotypic data (provided by LGC)
R
S
R
R
R
S
S
R
R
S
YOUR LOGO
?
S
S
S
S
R
?

13. Reliability evaluation of the selected markers, by comparison
between real phenotypes and phenotypes predicted by markers
Method :
Pheno. test
Genetic test
S = S
R = R
R ≠ S
or
S ≠ R
mismatch
R=R S=S R=R S=S S=S R=R R=R R≠S S=S R=R S=S R≠S
Reliability (%) = 100 - % mismatch
= 100- 2*100/12 = 83,3 %
FruitBreedomics Pilot studies results :
YOUR LOGO

14. Assessment and conclusions of the MAS on Rm2 gene
 10 markers were identified as good candidates for MAS on Rm2 gene from the (‘Pamirskij 5’
x ‘Rubira’®)2 (PR2) genetic map
 733 plants, coming from 6 populations, were genotyped with 2 markers selected among the
10 initially identified on the PR2 genetic map.
 Sample processing by LGC Genomics (extraction and genotyping) was fast enough (a few
days) and the data we received were of good quality (few missing data).
 The validation results with a test of resistance to the aphid on the 733 tested plants showed
about 90% of reliability (mismatch probably due to problems with phenotyping and/or leaf
sampling)
 Acquisition of good practices on leaf sampling in greenhouse and orchard
 Validation of a working METHODOLOGY to ensure the success of genetic testing of
progenies (screening first the polymorphism of genitors on the 10 markers identified from
the genetic map)
YOUR LOGO