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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
FB selected traits for whom MAB implémentation
is ongoing

Genetic linkage map of peach (8 chromosomes)

YOUR LOGO
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
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)
Development of a population (F2) segregating for
resistance to aphids

Genomic data
+
Phenotypic data

Genetic map

YOUR LOGO
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
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
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
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
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
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
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

?
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
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

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4 mab peach mauroux jehan baptiste

  • 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