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4 mab peach mauroux jehan baptiste

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  • 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