06 wp5 progresses&results-20130221
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06 wp5 progresses&results-20130221 06 wp5 progresses&results-20130221 Presentation Transcript

  • WP5 _ Enhancing the knowledge of genetics underlying novel traits and providing phenotyping methods Results achieved since the beginning of the project and plans for 2013
  • Main objectives of the WP5 • Develop tools for novel trait determination • Investigate novel and complex traits • Make novel trait analysis high throughput and applicable for the research community and the breeding industry
  • 3 tasks • Task 5.1 Improve Monilinia resistance in peach • Task 5.2 Assess fruit quality Jurriaan • Apple • Peach Remo • Task 5.3 Select traits important for climate change adaptation Evelyne
  • Results achieved since the beginning of the project Task 1. improve resistance to Monilinia in peach • Protocols for infection tests in lab • Test of artificial infections in orchard • Microscopy analysis of infection • Biochemical analyses of fruit surface • Tests of fungicide activity of some compounds • Spore survey in the orchard
  • Results achieved since the beginning of the project Task 1. improve resistance to Monilinia in peach Spray Monilinia laxa suspension Infection enhancement by humidity increasing: fruit covering One week incubation Susceptibility score: % infected fruits Setup of high-throughput orchard brown-rot phenotyping protocol CB2 CB3 inoculation covering no yes no C I paper CB2 IB2 plastic CB3 IB3
  • Results achieved since the beginning of the project Task 1. improve resistance to Monilinia in peach Spray Monilinia laxa suspension Infection enhancement by humidity increasing: fruit covering One week incubation Susceptibility score: % infected fruits Setup of high-throughput orchard brown-rot phenotyping protocol CB2 CB3 IB2 treatment allowed to distinguish between tolerant and susceptible accessions
  • cell collapse ( ) fungal colonization ( ) Monilinia disease progress : 8 vs. 48 hour after inoculation Zephir 8hpiZephir 48hpi E. Lady 8hpiE. Lady 48hpi Bolinha 8hpiBolinha 48hpi Bolinha: no fungal impact on the analysed samples
  • Results achieved since the beginning of the project Task 1. improve resistance to Monilinia in peach Biochemical analyses of fruit surface 40 60 80 100 120 140 160 050100150 Jours après floraison Massed'unfruit(g) A I II III 40 60 80 100 120 140 160 050100150 Jours après floraison Demi-circonférence(mm) B 40 60 80 0.00.51.01.52.0 Jours ap IndicedeDifférenced'Absorbance C Fruit growth for 2 cultivars Days after bloom Fruitmass(g) 020406080100 25-apr 16-may 30-may 20-jun 4-jul 18-jul maturity Infectionprobability(%) * ** * SG ZE Infection probability for 2 cultivars I II III
  • Results achieved since the beginning of the project Task 1. improve resistance to Monilinia in peach Cuticular conductance Biochemical analyses • surface compounds • waxes • cutins • epiderm and flesh phenolics 40 60 80 100 120 140 160 05101520 total cuticular wax quantities (mg/dm²) DAB waxaccumulationmg/dm² SG ZE Total cuticular waxes accumulation Wax(mg/dm²) Days after bloom 0 50 100 150 200 250 2004006008001000 masse du fruit (g) conductancedel'épiderme(cm/h) SG ZE Fruit mass (g) Cuticularconductance(cm/h) Cuticular conductance
  • Results achieved since the beginning of the project Task 1. improve resistance to Monilinia in peach Correlations between infection probability and surface compounds -1-0.75-0.5-0.2500.250.50.751 P Pic64.6_307 Pic65.7_312 -1 -0.75 -0.5 -0.25 Proportiond'infection Acideoléanolique Acideursolique Oleanolic acid Ursolic acid pcoumaroyl Pic52.5_312 Pic54.6_308 Pic64.6_307 Pic65.7_312 inhibitory candidates 020406080100 25-apr 16-may 30-may 20-jun 4-jul 18-jul maturity Infectionprobability(%) * ** * SG ZE Infection probability I II I I I mg/dm² 01234 Oleanolic acid mg/dm² 0246810 Ursolic acid Airedepic/dm²/10^5 051015202530 Pic52.5_312 Jours après floraison Airedepic/dm²/10^5 051015 Pic64.6_307 40 60 80 100 120 140 160 051015202530 Jours après floraison Airedepic/dm²/10^5 Pic65.7_312 40 60 80 100 120 140 160 0510152025 Airedepic/dm²/10^5 p-coumaroyl derivative SG ZE days after bloom Evolution of fruit surface compounds along fruit growth Days after bloom
  • Task5.2 Assess fruit quality State of progress
  • Research topic in task 5.2 • Texture/maturation/quality tools evaluation – Compare, use and evaluate common used methods (colour cards, pm, ss, tta, AWETA, DA) – Acoustic method for crispiness (FC, NZ) – Juice/juiciness analysis by punching and study tissue structure (NZ) – New methods for Fibre content (ML) • Gene expression based methods for analysing fruit texture trait – Ethylene pathway dedicated approach to unravel ripening and texture trait biomarkers (DLO) – Differential cultivars for harvest time, storage quality, shelf-life and meatiness (JP) • Storage stress test to select and early predict storage performance of new lines – Standard measurements and inspection (DLO) – Gene expression analysis (JP, DLO)
  • Texture/maturation/quality tools evaluation Quality measurements (n=20) • At harvest • After 1 week regime + 1 week shelf life (18°C, 75% RH) • After 1 month regime + 1 week shelf life (18°C, 75% RH) • After 2 months regime + 1 week shelf life (18°C, 75% RH) Monitored quality parameters: • Fruit weight • Ethylene production (at harvest) • Firmness (FTA automated penetrometer, destructive) • Firmness (AWETA acoustic, non-destructive) • Ground colour (colour chart) • Chlorophyll status (DA-meter, non-destructive) • External and internal disorders (expert visual evaluation)
  • SensorSense ETD 300 characteristics • Detection limit 300 ppt (0.3 ppb) • Upper limit 5 ppm (this one up to 100 ppm) • Measurement every 5 sec • Accuracy: <1% of 0.3 ppb (largest value) • Very specific for ethylene • Calibration yearly
  • 0 20 40 60 80 0 2 4 6 8 10 12 14 16 18 20 22 24 26 40 45 50 55 60 65 70 0 20 40 60 80 0 2 4 6 8 10 12 14 16 18 20 22 24 26 40 45 50 55 60 65 70 0 20 40 60 80 0 2 4 6 8 10 12 14 16 18 20 22 24 26 40 45 50 55 60 65 70 0 20 40 60 80 0 2 4 6 8 10 12 14 16 18 20 22 24 26 40 45 50 55 60 65 70 TA-XTplus - AED 4 mm flat head probe 5 Kg loading cell 100 mm/min Acquisition at 500 pps Compression strain: 90%
  • Approach Plant & Food Research • 6 cultivars • 10 trained panellists • 14 sensory texture attributes • 3 fruit per cultivar • 3 storage times (scheduled according to softening rate) • Air storage only • 1 harvest (using starch and skin colour
  • Approaches for improved phenotyping technologies – optimising existing technologies • Destructive • Tensile • Single edge notch beam • Rheometer (frequency sweep) • Juicer Kinetics • Microphone • Nondestructive • AWETA • Sinclair
  • Juiciness potential assoc. with larger cells, higher density and more apoplastic fluid Scifresh Sciros C .Pink R .G ala Sciearly C ox 0.75 0.80 0.85 0.90 0.95 Corticaldensity(g/mL) Scifresh Sciros RGala C.Pink 0 20000 40000 60000 80000 100000 Cellaream 2 Scifresh Sciros C .Pink R oyalgala 0.05 0.06 0.07 0.08 Apoplasticfluid(g/gFW)
  • List of tools Tas k Sub - tas k Sub-task name Trait or step Destructive / non Protocol / tool Tested (1 or 2 years) Species Conclusions on protocols available Feasibility Reliability High- throuput 2 2.1 Maturity assessment Index of differential absorbance IAD 1 apple not yet* y y y Starch D Y 2 " y y y y Solible solids D Y 2 " y y y y Titratable acids D Y 2 " y y y y Streif Index D Y 2 " y y y y Ethylene production N Y 1 " y y y y/n Colour determination N Y 2 " y y y y Chlorophyll ? status (DA-meter) N Y 1 " y y Not sure to what quality trait it links y
  • List of tools Tas k Su b- tas k Sub-task name Trait or step Sub-trait Protocol / tool Destructi ve / non Tested (1 or 2 years) Species Conclusions on protocols available Feasibilit y Reliabilit y High- throuput 2.2 Fruit texture assessment Physical paramet ers Firmness analysis by penetro measurements Texture Analyser D 1 apple y y y y hand-held penetromet er D x " x x <y x Firmness analysis by confined compression test D x " x x x x Firmness analysis by non- destructive acoustic resonance devices AWETA AFS N 1 " y y y y Sensor deform at impact sinclair y y y/n y Firmness analysis combined with crispness analysis acoustic- mechanical N x " x x x x Juice press/spin test D x " x x x x Color determination N 1 " y y y y Bioche mical paramet ers Soluble solids D 1 " y y y y/n Titratable acidity D 1 " y y y y/n Flavour x " x x x x Molecular characters of texture 1(sample s frozen) " n ? ? ?
  • Water contribution/microstructure on texture (tissue mechanical properties): Evaluation of NMR relaxometry as a screening tool Achieved •Water content & compartmentalization were accessed by relaxometry but were not directly related to mechanical properties •Freezing and thawing samples exacerbate relations between mechanical properties & relaxometric variables On going •Relate relaxometry & mechanical data with free sugar and cell wall compositions •New assays to evaluate relaxometry of frozen samples vs mechanical/histological/chemical variables New methods for quality screening assessment WP5.2 INRA-Nantes Biopolymers, Interactions, Assembly
  • Dietary fiber content and nature : development of fast screening method by Mid IR analysis of alcohol insoluble tissue material (AIM) Achieved •AIM, protein and Mid-IR of 29 genotypes On going •DF (AOAC method TDF), cell wall sugar analysis, ash and starch contents •Chemiometric analyses New methods for quality screening assessment WP5.2 INRA-Nantes Biopolymers, Interactions, Assembly
  • Identification of genes and networks controlling major apple quality traits Materials: 10 varieties (Golden, Gala, Elstar, Greensleave, Cox, Boskoop, Ariane, Jonagold, Fuji, Granny Smith) 4 contrasted hybrids 4 time points: Harvest, 1 month post-harvest, 2 MPH, 4 MPH 2 years of data: 2011&2012 Texture traits: Expert sensory panel (1 to 5 values): Fiber, Grain, Mealiness, Softness, Crunchiness, Juiciness, Acidity, Sugar Physical and Chemical measurements: Acidity, Sugar, Ethylene, Penetrometry, Compression Results: 2011: Phenotypic data completed (contrasted traits, differents kinetics of trait evolution) 2012: Finishing the 4MPH measurements RNA purification completed soon (112 samples) Transcriptomic results with the AryANE chip in June 2013 Bioanalayses and Network buildings during summer WP5-3 To be completed: material exchange with DLO and PFR (comparison with RNAseq) functional validation of the candidates
  • R scripts developed for data treatment 24 « .pair » files Raw data 12 « .txt » files Norm. data normalized intensities per sample, ratios, p.value, localFDR (Limma & fdrtool packages) RG_plot.png MA_plot.png PCAS Clusters Dendrograms ... complet-lists.txt Kjgqdkjlqk Kjgqdkjlqk Kjgqdkjlqk Kjgqdkjlqkghsfhf, Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk Kjgqdkjlqk Kjgqdkjlqk Kjgqdkjlqkghsfhf, Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk Kjgqdkjlqk Kjgqdkjlqk Kjgqdkjlqkghsfhf, Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk Kjgqdkjlqk Kjgqdkjlqk Kjgqdkjlqkghsfhf, Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk Kjgqdkjlqk Kjgqdkjlqk Kjgqdkjlqkghsfhf, Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk Kjgqdkjlqk Kjgqdkjlqk Kjgqdkjlqkghsfhf, Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk Kjgqdkjlqk Kjgqdkjlqk Kjgqdkjlqkghsfhf, Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Annotations.txt
  • MapMan Dongxia Yao et al Genomics Volume 98, Issue 1 2011 47 - 55
  • Storage Stress Test Tool • Success cultivar very much dependent on storage behaviour • Use commercial pick and commercial storage CA, DCS, mechanical/SF • Stress test and supporting tools to analyse this behaviour – Does it suffer in-cooling or CA stress – Is it Low Temp Sensitive – Firmness during storage – Sensitive to storage disorders
  • Experimental setup Five commercially grown cultivars 1. Cox O.P. 2. Elstar 3. Golden Delicious 4. Jonagold 5. Kanzi Harvested • a week before • at commercial harvest date (for long term ULO-storage) • 1 week after regime T (°C) %O2 %CO2 1 -1 21 0 2 -1 1 5 3 10 21 0 4 10 1 5
  • Overview of storage stress test (Y1) Cultivar Max storage period (optimal conditio ns) Low Temp Sensitivity flesh browning Texture issues Low O2/high CO2 tolerance Disorder sensitivity practic e test practice test practice test practice test Cox 5-6 m +++ Can develop mealiness before softening - Bitter pit, brown core, Softening Kanzi 12 m +++ Very firm, tough peel ? bitter pit, lenticel breakdown Jonagold 9 m - Greasiness ++ Scald, flesh browning Golden 8 m - ++ Scald Elstar 7 m + Rapid softening after storage + Softening, skin spots
  • Overview of storage stress test (Y1) Cultivar Max storage period (optimal conditio ns) Low Temp Sensitivity flesh browning Texture issues Low O2/high CO2 tolerance Disorder sensitivity practic e test practice test practice test practice test Cox 5-6 m +++ +++ (-1°C + CA, late harvest, ≥1 month) Can develop mealiness before softening Mealiness , rapid softening (air, ≥1 month) - - Bitter pit, brown core, Softening - Kanzi 12 m +++ ++++ (-1°C + CA, late harvest, 2 months) Very firm, tough peel Very firm indeed ? ++ (-1°C + CA, last harvest, ≥1 month) bitter pit, lenticel breakdown - Jonagold 9 m - - Greasiness Greasiness (10°C, air, ≥1 month) ++ + (-1°C + CA, 3 harvest, ≥1 month) Scald, flesh browning - Golden 8 m - - Greasiness (10°C, air, ≥1 month) ++ ++ (-1°C + CA, 1+3 harvest, ≥1 month) Scald - Elstar 7 m + - Rapid softening after storage Confirms practice + - Softening, skin spots Skin spots (all treatm)
  • Samples for microarray Cultivar Gen etic bac kgro und Max storage period (optimal conditions) Texture issues Low Temp Sensitivity flesh browning Low O2/high CO2 tolerance Disorder sensitivity practice test practice test practice test Cox 5-6 m Can develop mealiness before softening + +++ ++ - - Bitter pit, brown core, Softening Golden 8 m +/- - - ++ + Scald Kanzi 12 m Very firm, tough peel - +++ + ++(?) ++ bitter pit, lenticel breakdown
  • Task5.2 Assess fruit quality (peach) State of progress
  • Cultivar Texture Type Pulp Institution Cultivar Texture Type Pulp Institution UMIL IRTA ARO UMIL IRTA ARO Oro A NM Pc Y √ BO 05030142 SH P G √ BO 94007020 NM Pc Y √ BO 05030149 SH P G √ Dixired M P Y √ BO 05030081 SH P G √ Iride NM Pc W √ Elegant lady M P G √ Alice Col NM Pc Y √ IFF 331 SH P B √ Ambra M N Y √ √ Sweet dream M √ Big Top SM N Y √ √ Dulcebo SM P G √ Honey Kist SM N Y √ SRG M N G √ Redhaven M P Y √ Honey Royale SM √ Rich Lady SM P Y √ Nectaross M √ Vista Rich SM P Y √ BO 10120182 NM √ Ghiaccio SH P W √ √ Bolero M P G √ Glohaven M P Y √ BO 89010005 M N B √ Durado NM Pc Y √ Fei Cheng NM Pc B √ Alipersie M P Y √ Maria delizia M P B √ BO 00020006 NM Pc Y √ Dulciva M N G √ Swelling M P W √ Summer snow SM P W √ September Snow SM P W √ Fairlaine SM N Y √ 1881 SM P W √ Hermoza SM P W √
  • Plant material & Experimental Design Harvest ColdStorage(21dat4ºC) 14d 1d 3d 21+1d 21+3d 0d 21d 21+5d 5d Postharvest trial 2d 21+2d Classification M1 M2 M3 2011, 2012: M1, M2, M3
  • Summary (I) Trait Sub-trait Technique Years Comments Physical parameters Firmness analysis by penetro measurements Texture Analyser 1&2 Differentiate between fruit with different texture types through storage hand-held penetrometer 1&2 In some cases, differentiate between fruit with different texture types through storage Firmness analysis by confined compression test Texture Analyser 1 No additional information if compared to other physical firmness techniques Firmness analysis by non- destructive acoustic sensor AWETA AFS 1&2 Differentiate between stone fruit type but not different textures Compression to penetration ratio Texture Analyser 2 Interesting results in Y1 but not in Y2 Juice press & spin test Expressible juice 1&2 Differenciates fruit with different textures through storage Color determination & Portable spectrophotometer Minolta CR2600d 1&2 No suitable for differentiating fruit with different textures NIR Bruker MPA Multi Purpose FT-NIR Analyzer 0 Data currently being analyzed
  • Summary (II) Trait Sub-trait Technique Years Comments Biochemical parameters Soluble solids Standard 1&2 No relationship with texture Titratable acidity 1&2 Ethylene production 1&2 Not directly related to fruit texture, some interesting results that need to be further confirmed Antioxidant capacity FRAP assay 1&2 No direct relationship with fruit texture
  • Summary (III) Trait Sub-trait Protocol Years tested Comments Fruit structure and imaging TRS Instrument developed by Politecnico of Milano 1&2 Discriminate 3 out of 4 texture type (M, Sm and SH) Nuclear Magnetic resonance Esaote Airis II field intensity of 0,3 Tesla 1 No difference in texture (abandoned) Echography Multimage Aloka ssd-500 scanning frequency of 3,5-5,0-7,5 MHz 1 No difference in texture (abandoned) Computerized tomography Stratec Medizintechnik XCT Research SA+ 1&2 Gene expression RNA seq Illumina HiSeq 2000 2 Data currently being analysed Sensory evaluation Firmness perception 3 texture attributes and likeness 2 No capability to distinguish between texture types but differentiate through storage
  • Phenotyping tools  Standard quality (TSS, TTA)  Firmness (Penetrometry, Texture Analyser and Acoustic Firmness sensor)  Expressible juice  Objective colour (L*, a* and b*; Spectrophotometer 360-740nm)  Fruit ethylene production  Sensory Analysis (Consumer tests)  Biochemical analysis (Antioxidants, MDA…) Fmax F(5% Def.) P&D ratio F Stiffness Frecuency Impact Force
  • Phenotyping tools  Time Resolve Reflectance Spectroscopy (TRS)  Computerised tomography (CT)  Near Infrared Spectroscopy (NIR)  Echography  Nuclear Magnetic resonance(NMR)
  •  Gene expression analysis Transcriptomics cytokinesis cell stretching time Fase I Fase II Fase III Fase IV t0 t1 t2 t3
  • Results Fruit firmness: penetration Days at 20ºC 0 1 2 3 4 5 6 Firmness(Kg) 0 1 2 3 4 5 6 7 Days at 20ºC 0 1 2 3 4 5 6 Firmness(Kg) 0 1 2 3 4 5 6 7 Firmness(Kg) 0 1 2 3 4 5 6 7 Firmness(Kg) 0 1 2 3 4 5 6 7 Ambra (M)Firmness(Kg) 0 1 2 3 4 5 6 7 8 After CS Before CS Firmness(Kg) 0 2 4 6 8 Big Top (SM) Honey Royale (SM) Nectaross (M) Rome Star (M) Sweet Dream (SM) S M
  • -> No clear differences among different texture types -> Values from IRTA much lower than those from ARO (Impact of Agroclimatic conditions?) Am braB ig Top H oney R oyale N ectaross R om e Star Sw eetD reamO ded Sw elling 1881 Septem berSnow Fairlane (N ectarine) H erm oza Sum m erSnow P/Dratio 0 1 2 3 4 IRTA ARO Slow melting Melting Results Fruit firmness: deformation * * *
  • a a 0 10 20 30 40 50 60 70 80 0 1 2 3 4 5 ExpressibleJuice(%) Day at 20°C Melting Non Melting Slow Melting a a a b b b b bab c 0 10 20 30 40 50 60 70 80 0 1 2 3 4 5 ExpressibleJuice(%) Day at 20°C Melting Non Melting Stony Hard Slow Melting a a a a aab bc ab b b c b c b c 2012 2011 Results Expressible juice
  • Mie theory b s a    )(' Stony Hard Slow Melting Melting Results TRS
  • Mie theory b s a    )(' Stony Hard Slow Melting Melting Results TRS
  • Mie theory b s a    )(' Stony Hard Slow Melting Melting Results TRS
  • Mie theory b s a    )(' Stony Hard Slow Melting Melting Results TRS
  • Days at 20ºC 0 1 2 3 4 5 AcousticFirmness 0 10 20 30 40 50 Days at 20ºC 0 1 2 3 4 5 AcousticFirmness 0 10 20 30 40 50 AcousticFirmness 0 5 10 15 20 25 30 AcousticFirmness 0 5 10 15 20 25 30 AcousticFirmness 0 5 10 15 20 25 30 35 40 AcousticFirmness 0 5 10 15 20 25 30 35 40 After CS Before CS Ambra Big Top Honey Royale Nectaross Rome Star Sweet Dream Acoustic firmness changes did not reveal different softening behaviors among the different cultivars investigated Significant differences in the fruit acoustic firmness were observed between different stone fruit types (Nectarine vs Peach) Results Acoustic firmness Before cold storage After cold storage (21 d 4°C) (M) (M) (M) (SM) (SM) (SM)
  • Results CT
  • Results CT
  • Big Top Oro A Redhaven Alipersie Rich Lady IFF 331 BO 94007020 Ghiaccio SM M NM SH
  • 0 50 100 150 200 250 300 Canning peach Peach Nectarine Densitygcm-3 Texture within peach group Preliminary results average density 0 50 100 150 200 250 300 SM M SH Densitygcm-3
  • NGS raw data Quality trimming and filtering (erne-filter) Alignment to reference genome (Bowtie2) Hits raw count T1 T2 M SM SH NM M SM SH NM T1 M – Redhaven – Rep 1 1 0 0 0 0 0 0 0 M – Bolero – Rep 2 1 0 0 0 0 0 0 0 SM - Big Top – Rep 1 0 1 0 0 0 0 0 0 SM - Rich Lady – Rep 2 0 1 0 0 0 0 0 0 SH - IFF 331 – Rep 1 0 0 1 0 0 0 0 0 SH - BO05030081 – Rep 2 0 0 1 0 0 0 0 0 NM - Oro A – Rep 1 0 0 0 1 0 0 0 0 NM - BO010120182 – Rep 2 0 0 0 1 0 0 0 0 T2 M – Redhaven – Rep 1 0 0 0 0 1 0 0 0 M – Bolero – Rep 2 0 0 0 0 1 0 0 0 SM - Big Top – Rep 1 0 0 0 0 0 1 0 0 SM - Rich Lady – Rep 2 0 0 0 0 0 1 0 0 SH - IFF 331 – Rep 1 0 0 0 0 0 0 1 0 SH - BO05030081 – Rep 2 0 0 0 0 0 0 1 0 NM - Oro A – Rep 1 0 0 0 0 0 0 0 1 NM - BO010120182 – Rep 2 0 0 0 0 0 0 0 1 Differential expression analysis (R, Limma and EdgeR) Results Transcriptomic
  • For each type, the two time points group together. Only SH flesh type groups tightly together NM groups together at least in one axis, while M and SM samples group separately in both dimensions Results Transcriptomic
  • Conclusions and prespectives  It is feasible to differentiate between fruit with different textures using time-course postharvest experiments with certain techniques.
  • Task5.3 Adaptative traits State of progress INRA Montpellier and Bordeaux UNIBO
  • Objectives • Is it a genetic adaptation to ongoing climatic changes? • Setting protocols easy to perform on populations for genetic studies and to be duplicated in different sites • Chilling and heating requirements – Changes in temperature (during winter and spring) influences tree phenology – Can we phenotype for selecting cultivars with desired chilling and heating requirement ? • Water scarcity – Identification of physiological parameters, potential candidates for phenotyping tolerance and/or resilience to root water stress
  • Planned actions: CR et HR apple and peach • In apple: 3 cultivars (Golden Delicious, Gala, Granny Smith) • In peach: 9 cultivars (Fantasia, Ferjalou Jalousia, Flavorcrest, Mayglo, Redhaven, Summergrand, Summer Lady, Sunred, Tasty Free). chosen from bibliographical data on their respective temperature requirements (contrasting CR from 200 to 1000 CH). • Common methods performed in autumn 2011 and 2012 to estimate dates of dormancy release for floral and vegetative buds • Samples of shoots collected from October year n to february year n+1 (collection every week at each site from december to february) • Prospect other tests, search for new descriptors
  •  Two biological tests:  forcing of ‘one-bud cuttings’ (vegetative buds)  forcing of floral primordia (Tabuenca’s test) from paradormancy period (summer in year n-1) to ecodormancy period (winter year n)  Characterization of genetic and annual influences both in Southern France and Southern Brazil (bilateral Project Capes Cofecub) Observation of ‘green-tip’ stage: average time and percentage of budburst VEGETATIVE BUD FORCING FLORAL PRIMORDIA FORCING (within floral bud) Weighting of primordia before (in orchard ) and after forcing (fresh and dry weights) Results: CR et HR apple
  • Results CR in apple
  • 1 – Apple: Soil water restriction characterised by FTSW (Fraction of Total Soil Water) - Bologne: Same protocol than in 2011: Leaf T° and fluorescence - Mtp: with a volumetric control of water in the soil (Volumetric Humidity assessment experiment); * with mild root water restriction, applied during 3 weeks at 50% FTSW followed by 3 weeks at 20% FTSW, at morphological Measurements: (leaf area, shoot length and number of nodes) and eco- physiological (stomatal conductance) levels, • the ability to resume growth after a severe root water restriction, ie provoking apex growth arrest, and possibly death, for most genotypes, applied during 3, 4 and 5 weeks (resilience). 2 – Peach: 2 cvs grafted on a F1 rootstock progeny 2012 was the first year of water stress (field experiment) OBJECTIVES FOR WATER SCARCITY
  • 2. Identification of a cluster of physiological parameters, potential candidates for phenotyping Scatterplot (DATASHEET FATTORI pca STRESSED.sta 10v*17c) F2FLUOTL = -8.8167E-16+0.6582*x 7S 23S 26S35S 37S 38S 40S 41S 48S 54S 70S 96S 117S 121S 125S gsS stkS -4 -3 -2 -1 0 1 2 3 4 F2ALL -3 -2 -1 0 1 2 3 F2FLUOTL F2ALL:F2FLUOTL: r = 0.8153; p = 0.00007; y = -8.4004E-16 + 0.6582*x Scatterplot (DATASHEET FATTORI pca STRESSED.sta 10v*17c) F1FLUOTL = 6.1837E-16+0.6678*x 7S 23S 26S 35S 37S 38S 40S 41S 48S54S 70S96S 117S 121S 125S gsS stkS -6 -4 -2 0 2 4 6 8 10 F1ALL -4 -3 -2 -1 0 1 2 3 4 5 F1FLUOTL F1ALL:F1FLUOTL: r = 0.9387; p = 0.00000002; y = 4.3311E-16 + 0.6678*x PCA ALL vs. PCA FLUO_TL (2011)
  • 2. Identification of a cluster of physiological parameters, potential candidates for phenotyping PCA ALL vs. PCA FLUO_TL (2012) Scatterplot: F1_ALL vs. F1_FLUO_TL (Casewise MD deletion) F1_FLUO_TL = 0.0000 + .61268 * F1_ALL Correlation: r = .91001 7 23 26 35 37 38 40 41 48 54 57 70 96 106 111 117 121 125 gs stk -8 -6 -4 -2 0 2 4 F1_ALL -5 -4 -3 -2 -1 0 1 2 3 4 F1_FLUO_TL 95% confidence Scatterplot: F2_ALL vs. F2_FLUO_TL (Casewise MD deletion) F2_FLUO_TL = 0.0000 + .86070 * F2_ALL Correlation: r = .92362 7 23 26 35 37 3840 41 48 54 57 70 96 106 111 117 121 125 gs stk -3 -2 -1 0 1 2 3 4 F2_ALL -2 -1 0 1 2 3 4 F2_FLUO_TL 95% confidence
  • Projection of the cases on the factor-plane ( 1 x 2) Cases with sum of cosine square >= 0.00 Labelling variable: trt 7s 23s 26s 35s 37s 38s40s 41s 48s 54s 57s 70s 96s 106s 111s 117s 121s 125s GSs STKs -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 Factor 1: 53.19% -3 -2 -1 0 1 2 3 4 5 Factor2:31.55% Projection of the cases on the factor-plane ( 1 x 2) Cases with sum of cosine square >= 0.00 Labelling variable: trt Active 7S 23S 26S35S 37S 38S 40S 41S 48S 54S 70S 96S 117S 121S 125S gsS stkS -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 Factor 1: 65.47% -5 -4 -3 -2 -1 0 1 2 3 4 Factor2:24.93% 41 Stk 38 26 35 54 48 41 Gs 106 96 111 54 Gs 23 96 125 70 40 7 117 37 121 37 38 Stk 121 70 40 26 48 125 35 117 57 7 23 3. A tentative discrimination 2012 2011
  • As expected gs of 50% FTSW WS plants was higher than gs of 20% FTSW WS plants, with a decrease of ca. 80% and 35% compared to gs of WW plants, respectively. 7 23 26 35 37 38 4041 48 54 57 7096 97 106 111 117 121 125 GS SK 7 23 26 35 37 38 40 41 48 54 57 70 96 97 106 111 117 121 125 GS SK y = 0.1361x + 276.38 R² = 0.0464 ns y = 0.09x + 102.6 R² = 0.1802 P=0.05 0 50 100 150 200 250 300 350 400 450 0 100 200 300 400 500 600 2012-gsinWSplants(mmol.m-2.s-1) 2012- gs in WW plants (mmol.m-2.s-1) X12_gs_WS50 X12_gs_WS20 2012 results – Apple Mtp
  • BUT the ranking of genotypes relative to each other for the three traits in either WW or WS between the two periods was different (example of the number of nodes). This would suggest that apart from the proper effect of the water regime, a plant development effect (growth dynamics varying along the growing season) and/or other environmental factors likely different between the two measurement periods, affected the development of the shoot. Number of nodes developed during the period, either 50% or 20% Pearson coefficient / Rank correlation (Kendall’s t) WW - 50% FTSW vs WW - 20% FTSW R=0.32, ns; t=0.17, ns WS - 50% FTSW vs WS - 20% FTSW R=0.21, ns; t=0.15, ns 2012 results – Apple Mtp
  • The combination leaf temperature and fluorescence provides a very good compromise between rapid and effective assessment of the drought resistance of a given phenotype. WS protocol seems to be effective, simple for Leaf T° and fluorescence (and duplicable ??) Summary-APPLE Ranking was different between the two years and between stress conditions. Ongoing discussions and analyses
  • 0,0 5,0 10,0 15,0 20,0 25,0 0,0 20,0 40,0 60,0 80,0 100,0 120,0 140,0 1 2 3 4 5 6 7 8 9 10 11 12 temperature(°C) Rainfall(mm) month 2012 rainfall temperature 2012 Results on peach Harvest date Surprised: 19 and 23 July 2012 Summergrand: 31 July 2012 No rootstock effect on fruit growth But, fruit weight impacted … In the same year or from n-1 year effect?
  • Publications • Water scarcity – Unibo communication at ISHS symposium on « orchard management »in South Africa, Dec. 2012 – Joint statistical analyses between Unibo & Mtp: 1 common publication planned in 2013 • CR: – Statistical analyses of 2011 and 2012 results currently carriet out and publication planned, at least for apple results
  •  linked to other projects  technical improvements on forcing tests, and application to a segregant progeny (genetic determinism of chilling requirement trait)  Validation of chilling models previously selected in apple by AFEF Team, based on the forcing test results obtained in the two hemispheres  Apply of ‘one-bud cuttings’ test Research of new alternative methods as NIRS Technology, based on the forcing test results in France (French Project Perpheclim ACCAF) Main challenges for 2013 (CR, Apple)
  • Apple: 1 – Joint analyses between Bologna and Montpellier 2 - Same protocoles and observations in 2013, as in 2011 and 2012. No resilience experiment.  Need to better study the ranking of genotypes in the various water conditions and years 3 –Comparison of 1YO shoots between plants in a greenhouse and plants in nearby outside conditions in Montpellier + possibly, at least for some of the variables, comparison with adult-fruiting trees of the same genotypes in the field. Peach: New experiment in 2013 in Bordeaux (2nd year) and in Bologna (1st year) Main challenges for 2013 (Water scarcity)
  • Interactions WP5.3 and the rest of the project • Interactions with other WPs of the project: – Apply of ‘one-bud cuttings’ test to an apple segregant progeny (genetic determinism of chilling requirement trait) – Apply water restriction to an apple core collection from WP4
  • Publications • Communications in different meetings in 2012 and planned for 2013 • Publication plan has to be discussed during this meeting
  • Main challenges for 2013 • Complete sample analyses • Analyze all data acquired • Make synthetic analyses between years • Merge results between partners • Give conclusions on methods and tools • Complete D5.2 (due February 2013)
  • Interactions between WP5 and the rest of the project • From WP5 to WP2, WP3, WP4 – phenotyping tools for WP2, WP3 and WP4 Ex: Monilinia resistance on peach ; crispiness and water scarcity on apple – provide interesting genitors to WP2 • Interactions planned with the stakeholders of the project Test the phenotyping protocoles when available
  • Action Plan for 2013 To be discussed during this meeting Should include many exchanges between partners via mels, phone calls, visio conf or workshop meetings
  • WP5 workshops This afternoon: • Task 5.2 _ 15:30 to 18:30 _ sala delegacions • Task 5.3 _ 17:00 to 18:30 _ room 2.06 Tomorrow morning: 8:30 to 10 : 3 tasks separately