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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

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FB selected traits for whom MAB implémentation is ongoing

Genetic linkage map of peach (8 chromosomes)

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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.

Real world application of markers in peach breeding programs: Pilot studies on Rm2

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Implementation of MAS for the resistance of green aphid : from gene mapping to marker validation

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)

Marker

Identification

• From the resulting map, identification of a set of markers close to the Rm2 gene

Marker

Checking

•Leaf sampling

•Genotyping parents with the 10 previously identified markers

•Screening of the set of markers on the parents of 6 targeted crosses

Genetic Test on

Offsprings

•Leaf sampling

•Genotyping individuals with the 2 selected markers

•Establishment of a list of resistant and susceptible individuals

Marker

Validatio

n

•Reliability evaluation of the selected markers, by comparison between real phenotypes and phenotypes predicted by markers

Mar

ker d

evel

opm

ent

MAB

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Genetic map

Genomic data+

Phenotypic data

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

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Without markers

Methodology for testing resistance to green peach aphid

Resistance testAphid production

Multiplication(on GF305)

Controlled infestation +Phenotyping

Mass rearing(on 3 month seedlings)

Aphids transfert

Susceptibles

Resistants

1 week

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Using markers

Methodology for testing resistance to green peach aphid

Resistance testAphid production

Multiplication(on GF305)

Controlled infestation +Phenotyping

Mass rearing(on 3 month seedlings)

Aphids transfert

Markers

Markers

Susceptibles

Resistants

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.

1 week

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Creation of a genetic map (markers + Rm2)

CH1 CH2 CH3 CH4 CH5 CH6 CH7 CH8

Rm2

Genetic map derived from the (Pamir x Rubira)2 cross

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From the resulting map, identification of a set of markers close to the Rm2 gene

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

Chromosome 1

Zoom +

End of the chromosome 1

mkr01mkr02mkr03mkr04mkr05mkr06mkr07mkr08mkr09mkr10

Rm2

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Leaf sampling + Genetic Tests

Polymorphic marker Monomorphic markeror

R m2

Screening of the set of markers on the parents of 6 targeted crosses

Parent of the pilot studies:

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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

Leaf sampling

- DNA extraction- Genotyping

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Exemple of genotypic data (provided by LGC)

R S S SR R R R R R ?S S S S S?

Establishment of a list of resistant and susceptible individuals

R m2 R m2 R m2

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Genetic testPheno. test

S = S R = R

R ≠ Sor

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 %

Method :

FruitBreedomics Pilot studies results :

Reliability evaluation of the selected markers, by comparison between real phenotypes and phenotypes predicted by markers

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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)

Assessment and conclusions of the MAS on Rm2 gene