Published on February 28, 2014
Pilot studies on Marker Assisted Breeding: Apple Markus Kellerhals, Lucie Leumann, Simone Schütz, Isabelle Baumgartner, Andrea Patocchi, Agroscope YOUR LOGO
Apple breeding at Agroscope: selection steps seedlings Crosses (MAPS) 10 000 2 years 1 Selection in glasshouse (MASS) and container seedling nursery (MASS) Grafting on M27 with interstem 4000 4-8 Step 1: 1 tree on M27 600 8-12 Stage A: 3 trees on M9 30 10-17 Stage B: 4 x 4 trees on M9 4 14-20 Stage C: 50 trees, 1 row, on M9 2 3 600 Commercialisation: 1 variety out of 30 000 to 50 000 seeds after 14 to 20 years YOUR LOGO
Marker application in apple breeding Fruitbreedomics WP1: Increasing efficiency of (marker-assisted) breeding (MAB) of new cultivars Pilot studies to check the practical application of MAB: Agroscope (CH) and University Bologna (I) Use of newly developed SNP (Single Nucleotide Polymorphism) markers instead of SSR (Simple Sequence Repaeat) markers
Early selection towards pyramided disease resistances and fruit quality Disease resistance Scab (V. inaequalis) Major resistance genes: Rvi6 (Vf), Rvi2 (Vh2) Mildew (P. leucotricha) Major resistance gene: Pl2 Fruit quality Crispness, texture, acidity Shelf life, storage (ethylene)
Pilot Studies: Crosses at Agroscope Parents were analysed with 20k SNP chip at Wageningen UR Mother Father Flowers Fruits Seeds ACW 11303 (Rvi6=Vf) ACW 18522 (Rvi6=Vf, Rvi2=Vh2) 2246 352 2227 ACW 13652 (Rvi6=Vf, Pl2) ACW 11567 (Rvi2=Vh2) 1370 323 2793
Steps Breeding strategy developed and crosses made (2011) New information on false positive reactions with some molecular markers (SSR marker present but gene not) led to adaptation of the strategy Sowing and screening for scab resistance (phenotypic), spring 2013, susceptible plants removed (except in subpopulations) Molecular selection with SNP’s (June 2013) Potting of selected plants for container field (July 2013) Phenotypic selection of the potted plants (October 2013) Grafting for selection field and for second scab screening in the glasshouse (second screening in 2015)
Crosses and glasshouse screening
Sampling for molecular analysis - No lyophilisation necessary - Deep well block, put the leaf rondelle, a plastic film and silica gel
Markers used Trait / Locus Marker Scab resistance Rvi6=Vf (LG1) Rvi6_42M10SP6_Y124 (SNP) Scab resistance Rvi2=Vh2 (LG2) Rvi2_region53_M417 (SNP) Mildew resistance Pl2 (LG11) Pl2_3_Y211 (SNP) Fruit texture Md-PG1 (LG10) PG_FEM_LC_19 Acidity (LG16) Acidity_SNP2 (ss475876558; SNP2 from RosBREED) Crispness (LG16) Crispness_SNP1 (ss475881704; SNP1 from RosBREED) Shelf life Md-ACO1 (LG10) ACO_FEM_cg_4 Shelf life Md-ACS1 (LG15) ACS_FEM_cg_5
MAB experiences • Plates of 6 x 4 pots allowed plant identification without labelling • Missing plants were replaced by living plants to fill up gaps in the plates (cost), laborious • Puncturing the leaves and expedition of the plates was efficient • Parents had to be checked first for the polymorphism of the planned SNP markers • The whole procedure was more time-consuming than expected • Costs are relatively low, DNA extraction is the most expensive, data point to low price • Close interaction with the company KASP - SNP Genotyping (LGC genomics) was required and successful
Analyses with LGC Genomics (UK) • Analysis of 2500 progeny plants (1250 from each progeny) financed by Fruitbreedomics • Analysis of remaining progeny plants financed by Agroscope (useful backup pool)
Pilot studies ‘Fruitbreedomics’ Individual plant Data sheet with the results of molecular analysis Rvi6_42M10SP6_Y124 T = Vf resistance Cross, plate position MasterPlate MasterWell Call 1206-5 H06 C:T 1206-5 D09 C:T 1206-5 G09 C:T 1206-5 F10 C:T 1206-5 G10 C:T 1206-5 C12 C:T 1206-6 B01 C:T 1206-6 H01 C:T 1206-6 F02 C:T 1206-6 G02 C:T 1206-6 A03 C:T 1206-6 H04 C:T 1206-6 E05 C:T 1206-6 G05 C:T 1206-6 H05 C:T Pl2_3_Y211 T = PL2 resistance Marker SNPID MasterPlate MasterWell Call Rvi6_42M10SP6_Y124 1206-5 H06 C:T Rvi6_42M10SP6_Y124 1206-5 D09 C:T Rvi6_42M10SP6_Y124 1206-5 G09 C:T Rvi6_42M10SP6_Y124 1206-5 F10 C:T Rvi6_42M10SP6_Y124 1206-5 G10 C:T Rvi6_42M10SP6_Y124 1206-5 C12 C:T Rvi6_42M10SP6_Y124 1206-6 B01 C:T Rvi6_42M10SP6_Y124 1206-6 H01 C:T Rvi6_42M10SP6_Y124 1206-6 F02 C:T Rvi6_42M10SP6_Y124 1206-6 G02 C:T Rvi6_42M10SP6_Y124 1206-6 A03 C:T Rvi6_42M10SP6_Y124 1206-6 H04 C:T Rvi6_42M10SP6_Y124 1206-6 E05 C:T Rvi6_42M10SP6_Y124 1206-6 G05 C:T Rvi6_42M10SP6_Y124 1206-6 H05 C:T SNPID Pl2_3_Y211 Pl2_3_Y211 Pl2_3_Y211 Pl2_3_Y211 Pl2_3_Y211 Pl2_3_Y211 Pl2_3_Y211 Pl2_3_Y211 Pl2_3_Y211 Pl2_3_Y211 Pl2_3_Y211 Pl2_3_Y211 Pl2_3_Y211 Pl2_3_Y211 Pl2_3_Y211
Strategy and Reality cross 1 cross 1: ACW 11303 (Rvi6) x ACW 18522 (Rvi6, Rvi2): keep 50 random plants, keep 50 scab susceptible plants (class 4), keep all plants with two or more resistances according to molecular analyses (take out of the resistant part 50 without fruit quality-markers) Segregation expected and observed (based on the marker analysis) Rvi6Rvi6Rvi2 observed % observed expected from 1250 % expected Selected for grafting Rvi6Rvi6 Rvi6Rvi2 Rvi6 Rvi2 no resistance total 67 235 298 258 164 75 1097 6.1 % 21.4 % 27.2 % 23.5 % 15.0 % 6.8 % 100 % 156 156 312 312 156 156 1250 12.5 % 12.5 % 25.0 % 25.0 % 12.5 % 12.5 % 100 % 31 5 93 8 10 29 176
Strategy and Reality cross 2 cross 2: ACW 13652 (Rvi6, Pl2) x ACW 11567 (Rvi2): keep 50 random plants, keep 50 scab susceptible plants, keep all plants with three resistances according to molecular analyses Rvi2Rvi6Pl2 observed observed % expected from 1250 % expected Selected for grafting Rvi2 Rvi6 Rvi6 Pl2 Rvi2 Pl2 Pl2 Rvi6 Rvi2 no total resistance 147 66 1061 13.8 % 6.2 % 133 12.5 % 148 13.9 % 162 15.2 % 150 14.2 % 71 6.7 % 184 17.3 % 156 156 156 156 156 156 156 156 1250 12.50% 47 12.50% 2 12.50% 5 12.50% 6 12.50% 7 12.50% 3 12.50% 11 12.50% 19 100% 100
Envisaged further selection Jan 2014 Feb 2014 or 2015 Winter/Spring 2015 Jun/July 15 Jun/July 16 grafting on rootstock M 27 with intermediate ‘Schneiderapfel’ planting to level 1 second glasshouse inoculation with different scab strain first assessment for fruit quality (about 30-40% fruiting) and scoring of tree characteristics first major assessment for fruit quality (about 80% fruiting), tree scoring, first comparison of fruit quality and marker results for fruit quality, comparison of tree characteristics and marker set, powdery mildew in cross 2
Conclusions MAB is a useful tool to increase efficiency in fruit breeding Careful parent selection and molecular characterization is important to avoid misinterpretation Check with the phenotype Close interaction with the company which analyses the samples
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