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Pilot studies for efficient early phenotypic and molecular selection for disease resistance and fruit quality traits in apple

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

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 Repeat) markers

Marker assisted apple breeding

1 variety with commercial potential 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: 4 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

Pilot studies integrated in the selections steps of the Agroscope apple breeding program

Mother (Resistances) Father (Resistances) 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

Pilot Studies: Crosses at Agroscope (2011)

Progeny 1

Progeny 2

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)

Early selection towards pyramided disease resistances and fruit quality

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 (SNP)

Shelf life Md-ACO1 (LG10) ACO_FEM_cg_4 (SNP)

Shelf life Md-ACS1 (LG15) ACS_FEM_cg_5 (SNP)

Crispness (LG16) Crispness_SNP1 (ss475881704; SNP1 from RosBREED)

Acidity (LG16) Acidity_SNP2 (ss475876558; SNP2 from RosBREED)

Markers used

Breeding/selection strategy developed and two crosses made (2011) New information on false positive reactions with some molecular markers (SSR

marker present but gene not) in the parents led to adaptation of the strategy Sowing and screening for phenotypic scab resistance (spring 2013), susceptible

plants removed (except in subpopulations) Molecular analysis with SNP’s (June 2013) Potting of selected plants based on resistance for container plot (July 2013) Phenotypic selection (juvenility, mildew) of the potted plants also considering

breeding strategy and the molecular analysis (October 2013) Grafting one tree per genotype for fruit selection in the orchard and one tree per

genotype for the test of the scab resistance pyramid (inoculation with Rvi6-virulent strain in the glasshouse in 2015)

Planting to orchard (spring 2015)

Steps

Crosses and glasshouse scab screening

Progeny 1 Progeny 2

Polymorphism of the SNP alleles checked in the parents No lyophilisation necessary Deep well block, put the leaf rondelle, a plastic film and silica gel Shipment to LGC Genomics for analysis

Sampling leaf material for molecular analysis (SNP’s)

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

Seedling selection

Rvi2Rvi6Rvi6 Rvi2Rvi6 Rvi2 Rvi6Rvi6 Rvi6 no resistance total

Observed (N) 67 298 164 235 258 75 1097

Observed (%) 6.1 % 27.2 % 15.0 % 21.4 % 23.5 % 6.8 % 100 %

Expected from 1250 (N) 156 312 156 156 312 156 1250

Expected (%) 12.5 % 25.0 % 12.5 % 12.5 % 25.0 % 12.5 % 100 %

Selected for grafting (N) 31 93 10 5 8 29 176

cross 1: ACW 11303 (Rvi6) x ACW 18522 (Rvi6, Rvi2) keep 50 random plants keep 50 scab susceptible plants (phenotyping class 4) keep all plants with two resistances according to molecular analyses (subset: out of the resistant part 50 with undesired fruit quality-marker combinations)

Segregation expected and observed (based on the molecular analysis)

Strategy and Reality cross 1

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

Strategy and Reality cross 2

Segregation expected and observed (based on the molecular analysis) Rvi2Rvi6Pl2 Rvi2Rvi6 Rvi2Pl2 Rvi6Pl2 Rvi2 Rvi6 Pl2 no resistance total

Observed (N) 133 148 150 162 147 184 71 66 1061

Observed (%)12.5 % 13.9 % 14.2 % 15.2 % 13.8 % 17.3 % 6.7 % 6.2 %

Expected from 1250 (N)

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 (N)

47 2 6 5 11 3 7 19 100

PG_FEM_LC_19 Crispness_SNP1 Acidity_SNP2 avoid T T:T less crisp than T:C,

C:C bestA:A more acid than C:C, A:C intermediate

ACW 11303 C:C T:C intermediate A:C intermediate

ACW 18522 T:C C:C best A:A more acid

Prefered genotypes (%)in progeny

C:C (46%) C:C best (52%) A:C intermediate (48%)

Segregation of fruit quality markers

PG_FEM_LC_19 ACO_FEM_cg_4 ACS_FEMcg_5 Crispness_SNP1 Acidity_SNP2

avoid T -> not possible

G = good A = good,A:A best

T:T less crisp than T:C, C:C best

A:A more acid than C:C, A:C intermediate

ACW13652 T:C A:G good A:A best T:T less crisp A:C intermediate

ACW11567 T:T A:A A:G good T:C intermediate A:C intermediate

Prefered genotypes (%) in progeny

T:C (50%) A:G good (51%) A:A best (44%) T:C intermediate (54%)A:C intermediate (50%)

cross 1: ACW 11303 (Rvi6) x ACW 18522 (Rvi6, Rvi2)

cross 2: ACW 13652 (Rvi6, Pl2) x ACW 11567 (Rvi2)

(Four) 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

MAB experiences

Container plot

From 5020 seeds 350 selections planted as grafted trees to selection step 1 orchard (spring 2015)

2 series (1205: March 2015, 1206: April 2015) Inoculum: Rvi6 (Vf)-virulent strain, Isolate D42, spray

concentration: 1.35 x 105 sp/ml (series 1), 2.2 x 10 105 sp/ml (series 2) in security glasshouse level 2

germination rate 24h: 68% (series 1), 75% (series 2)

Germinated conidia

Stellate necrosis: typical for Rvi2 scab resistancePlants with tent (humidity > 90%)

Test of the scab resistance pyramid (spring 2015)

Scab class / symptom 0 1 2 3a 3b Stellate

necrosisStellate

chlorosis 0 - 3b 4

Scab res. gene combination

Number of plants (percent of total plants)

Rvi2, Rvi6 28 86% 0% 14% 0% 0% 7% 4% 100% 0%

Rvi2 9 44% 0% 56% 0% 0% 33% 11% 100% 0%

Rvi6 11 0% 0% 9% 0% 0% 9% 0% 9% 91%

none 14 0% 0% 7% 0% 0% 0% 7% 7% 93%

Scab class / symptom 0 1 2 3a 3b Stellate

necrosisStellate

chlorosis 0 - 3b 4

Scab res. gene combination

Number of plants (percent of total plants)

Rvi2, Rvi6 26 58% 0% 42% 0% 0% 42% 0% 100% 0%

Rvi2 12 25% 0% 67% 8% 0% 75% 8% 100% 0%

Rvi6 4 0% 0% 0% 0% 0% 0% 0% 0% 100%none 16 0% 0% 0% 0% 0% 0% 0% 0% 100%

Results of scab inoculation with Rvi6-virulent strain

cross 1: ACW 11303 (Rvi6) x ACW 18522 (Rvi6, Rvi2)

cross 2: ACW 13652 (Rvi6, Pl2) x ACW 11567 (Rvi2)

autumn 2016 first assessment for fruit quality (about 30-40% fruiting) scoring of tree characteristics

autumn 2017 major assessment for fruit quality (about 80% fruiting) scoring of tree characteristics comparison of phenotypic fruit quality and

marker results for fruit quality traits cross 2: comparison of phenotypic powdery

mildew resistance with Pl2 marker results

Further steps

Plant plate to extraction plate logistics and plant identification throughout the whole process needs further improvement

MAB for disease resistance loci is more advanced than for fruit quality traits

Careful parent selection and molecular characterization (check for false positive and polymorphism of the markers) is important to avoid misinterpretation

Check with the phenotype (scab pyramid, fruit quality, etc.) Close interaction with the company which analyses the samples The defined strategy could not be 100% realised MAB is a useful tool to increase efficiency in fruit breeding

Conclusions

Acknowledgements

The team Financial support

Fruit-growing team Agroscope Cameron Peace, WSU FEM WUR for the scab isolate