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Pilot studies on Marker Assisted Breeding: Apple

Markus Kellerhals, Lucie Leumann, Simone Schütz, Isabelle Baumgartner, Andrea Patocchi, Agroscope

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Apple breeding at Agroscope: selection steps

Commercialisation: 1 variety out of 30 000 to 50 000 seeds after 14 to 20 years

year

s

Stage C: 50 trees, 1 row, on M9

Stage B: 4 x 4 trees on M9

Stage A: 3 trees on M9

Step 1: 1 tree on M27

Grafting on M27 with interstem

Selection in glasshouse (MASS) and container seedling nursery (MASS)

Crosses (MAPS)

14-20

10-17

8-12

4-8

3

2

1

2

4

30

600

600

4000

10 000

seedlings

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 resistanceScab (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

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

Parents were analysed with 20k SNP chip at Wageningen UR

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(LGC genomics) was required and successful

KASP - SNP Genotyping

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’

Rvi6_42M10SP6_Y124 Pl2_3_Y211Cross, plate position T = Vf resistance Marker T = PL2 resistanceMasterPlate MasterWell Call SNPID MasterPlate MasterWell Call SNPID1206-5 H06 C:T Rvi6_42M10SP6_Y124 1206-5 H06 C:T Pl2_3_Y2111206-5 D09 C:T Rvi6_42M10SP6_Y124 1206-5 D09 C:T Pl2_3_Y2111206-5 G09 C:T Rvi6_42M10SP6_Y124 1206-5 G09 C:T Pl2_3_Y2111206-5 F10 C:T Rvi6_42M10SP6_Y124 1206-5 F10 C:T Pl2_3_Y2111206-5 G10 C:T Rvi6_42M10SP6_Y124 1206-5 G10 C:T Pl2_3_Y2111206-5 C12 C:T Rvi6_42M10SP6_Y124 1206-5 C12 C:T Pl2_3_Y2111206-6 B01 C:T Rvi6_42M10SP6_Y124 1206-6 B01 C:T Pl2_3_Y2111206-6 H01 C:T Rvi6_42M10SP6_Y124 1206-6 H01 C:T Pl2_3_Y2111206-6 F02 C:T Rvi6_42M10SP6_Y124 1206-6 F02 C:T Pl2_3_Y2111206-6 G02 C:T Rvi6_42M10SP6_Y124 1206-6 G02 C:T Pl2_3_Y2111206-6 A03 C:T Rvi6_42M10SP6_Y124 1206-6 A03 C:T Pl2_3_Y2111206-6 H04 C:T Rvi6_42M10SP6_Y124 1206-6 H04 C:T Pl2_3_Y2111206-6 E05 C:T Rvi6_42M10SP6_Y124 1206-6 E05 C:T Pl2_3_Y2111206-6 G05 C:T Rvi6_42M10SP6_Y124 1206-6 G05 C:T Pl2_3_Y2111206-6 H05 C:T Rvi6_42M10SP6_Y124 1206-6 H05 C:T Pl2_3_Y211

Data sheet with the results of molecular analysis

Individual plant

Strategy and Reality cross 1

Rvi6Rvi6Rvi2 Rvi6Rvi6 Rvi6Rvi2 Rvi6 Rvi2 no resistance total

observed 67 235 298 258 164 75 1097

% observed 6.1 % 21.4 % 27.2 % 23.5 % 15.0 % 6.8 % 100 %

expected from 1250

156 156 312 312 156 156 1250

% expected 12.5 % 12.5 % 25.0 % 25.0 % 12.5 % 12.5 % 100 %

Selected for grafting

31 5 93 8 10 29 176

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)

Strategy and Reality cross 2

Rvi2Rvi6Pl2 Rvi2Rvi6

Rvi6Pl2

Rvi2Pl2

Pl2 Rvi6 Rvi2 no resistance

total

observed 133 148 162 150 71 184 147 66 1061

observed %12.5 % 13.9 % 15.2 % 14.2 % 6.7 % 17.3 % 13.8 % 6.2 %

expected from 1250

156 156 156 156 156 156 156 156 1250

% expected 12.50% 12.50% 12.50% 12.50% 12.50% 12.50% 12.50% 12.50% 100%Selected for grafting

47 2 5 6 7 3 11 19 100

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

Envisaged further selection

Jan 2014 grafting on rootstock M 27 with intermediate ‘Schneiderapfel’

Feb 2014 or 2015 planting to level 1 Winter/Spring 2015 second glasshouse inoculation with different scab

strainJun/July 15 first assessment for fruit quality (about 30-40%

fruiting) and scoring of tree characteristicsJun/July 16 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

Plant identification

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