4 mab peach mauroux jehan baptiste
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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
YOUR LOGO
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