Published on February 28, 2014
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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