WP1

Results achieved since the beginning of the project and plans for 2013

The general objective of WP1

… is to develop more efficient breeding programs applying MAB. To reach this goal WP1 will design and develop: • Selection strategies and schemes to be tested in

pilot studies • SNP markers tightly linked to selected genes will

be developed • Cost efficient MAB pipelines will be identified and

used to genotype the plant material of the pilot studies

State of progress of the ongoing tasks (in a nutshell)

• Task 1.1: Breeding strategies (AIM: understand how apple & peach a breed and develop better breeding strategies

– First deliverable of the WP (D1.1: description of ideotypes and breeding schemes) submitted at M18

– Applicability of GWS in apple and peach as been studied and found to be possible (see also experiments at PFR)

• Task 1.2: development/identification of SNP markers for genes mapped before the start of the project

– Apple: SNP makers identified for 12 out 18 loci

– Peach SNP makers identified for 7 out 12 loci

• Task 1.3: Pilot studies (AIM: demonstrate the power of MAS)

– Prepared list of PSP for both apple and peach, for peach final list in preparation

– GWS for apple planned (peach in consideration): populations identified, genotyping with 384 SNPs is sufficient; genotyping approach and company identified, financing is possible within partner own budget!

• Task 1.4: Genotyping pipeline (AIM: apply MAS using T.1.2 SNPs to PSP)

– In the process of finalizing MAB protocol (deadline M24!)

– In collaboration FEM (WP6) offers from genotyping companies have been collected and a company with a good offer has been identified

• Task 1.5: Specification list of breeder-interface (AIM: develop an breeder-friendly interface to query the DB)

– No much progresses have been made in this task. As deadline (M30!) is approaching activities have to start during this meeting and continue until September

(Main) results achieved so far

• Task 1.1: Breeding strategies – Thanks two stakeholder surveys we were able to figure out how apple

and peach are currently selected and which are the traits most important for apple and peach breeders • Apple results: see François talk of the stakeholder day 2012 • Peach results: see Thierry talk (in few minutes)

• Task 1.2: SNP markers – Apple (12/18); SNP makers identified for: Rvi2 (Vh2), Rvi4 (Vh4), Rvi5

(Vm), Rvi6 (Vf), Rvi11 (Vbj), Rvi13 (Vdurello), Rvi15 (Vr2), QTLs LG17 Fiesta and Discovery, FB-MR5, FB-E and Pl2 (nearly finish Rvi12 (Vb)) • Examples of the results and surprises: see Melanie Jänsch (talk in few

minutes)

– Peach (7/12); SNP markers identified for: Rm2, Vr2, D, S, G, Y and F

Publications

• 1 published scientific paper:

• Kumar et al. 2012: Genomic Selection for Fruit Quality Traits in Apple(Malus x domestica Borkh.). PLoSONE 7(5): e36674.

• But in 2013 (M28) we have D1.4 which is composed by the submitted manuscripts reporting the fine mapping and development of SNP markers for selected major genes and QTLs (output of T1.2)

Main challenges for 2013

ALL Deliverables and milestones to be done in the next 6 months (chronological order): • D1.4 (M28): submitted manuscripts on fine mapping of selected major

genes and QTLs (output of T1.2) • D1.2 (M30): Evaluation of breeding strategies used in other crops (GWS

included) (output of T1.1)

• MS4 (M24 not 28): markers tightly linked to the loci of interest (report) • MS5 (24) Optimized MAB protocol (report) • MS3 (M30): Evaluation of breeding strategies used in other crops

(review) • MS6 (M30): Specifications of breeder interface defined (report)

Action Plan for the coming year

WHAT WHO HOW DEADLINE

T1.1 submit D1.2 “Evaluation of breeding strategies used in other crops (GWS included)”

DLO According partner’s strategy

M30

T1.2 finish SNP identification and submit D1.3 (MS on fine mapping of selected major genes and QTLs)

EVD, DCA, JKI, IRTA, UMIL, INRA

According partner’s strategies

M28

T1.3 perform MAB in apple and peach; Perform GWS in apple

MAB: Apple and peach breeders involved in the task; GS: B3F, Novadi with the support of INRA and DLO

Breeders make plant material available; Statistical support for GWS by INRA/DLO

If feasible before harvest in order to identify the material to phenotype

T1.4 Test genotyping platform Genotype pilot studies populations

Partners involved and selected company

According protocols

M25 M30

T1.5 specification list breeder-interface ALL Being active and reactive to DCA requests

M30

Interactions between WP1 and the rest of the project

• Interactions planned with WP3, 4 and 7 (breeder interface)

• Interactions planned with the stakeholders:

– None planned

Selected short talks

• Melanie Jänsch

Rvi2, Rvi4 and Rvi11: SNP markers and surprises

• Thierry Pascal

Peach part of Deliverable 1.1

Rvi2, Rvi4, Rvi11: SNP markers and surprises

Melanie Jänsch, Juliane Weger, Giovanni Broggini, Vincent Bus, Sue Gardiner, Heather Bassett and Andrea Patocchi

Objectives

1. Identification of 5 single nucleotide polymorphisms (SNPs) within an interval of 1 cM

2. Check for level of polymorphism of SNPs in 8 founders

3. Focus of this talk: apple scab resistance genes Rvi 11 (Vbj), Rvi 2 (Vh2) and Rvi 4 (Vh4)

(Bus et al., 2011)

repeat

Method – SNP development

or reject region

BLAST available flanking markers on GD sequence

Primer Design + PCR

SEQ and SNP analysis SNP found

SNP confirmation of map position with subset of resistant , susceptible and recombinant individuals closest to the gene

SNP polymorphism test with 8 founders

SNP identified and validated

ok

ok

ok

ok

reject region

reject region

SNP polymorphism example - Rvi4 (Vh4)

250 260 270 280 290 300 310 320 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| 2249_TNL1 CGTGTTTCTGAAATGCTTCAGCTAAATCTCCGTCCTGCTTCCTGACATGCGAAGGATCAACGTGATAGAATATTGGCAMA Gala_TNL1 CGTGTTTATGAAATGCTTCGGCAAAATCTCCGTCCTGCTTCCTGATATGCGAAGGATCAACGTGATAGAATATTGGCAAA Braeburn_TNL1 CGTGTTTCTGAAATGCTTCGGCTAAATCTCCGTTCTGCTTCCTGATATGCGAAGGATCAACGTGATAGAATATTGGCAAA Cox_TNL1 CGTGTTTCTGAAATGCTTCGGCTAAATCTCCGTTCTGCTTCCTGATATGCGAAGGATCAACGTGATAGAATATTGGCAAA Delicious_TNL1 CGTGTTTCTGAAATGCTTCGGCTAAATCTCCGTTCTGCTTCCTGATATGCGAAGGATCAACGTGATAGAATATTGGCAAA F2_TNL1 CGTGTTTCTGAAATGCTTCGGCTAAATCTCCGTTCTGCTTCCTGATATGCGAAGGATCAACGTGATAGAATATTGGCAAA Golden_TNL1 CGTGTTTATGAAATGCTTCGGCAAAATCTCCGTCCTGCTTCTTGACATGCGAAGGATCAACGTGATAGAATATTGGCAAA Granny_TNL1 CGTGTTTCTGAAATGCTTCGGCTAAATCTCCGTTCTGCTTCCTGATATGCGAAGGATCAACGTGATAGAATATTGGCAAA Jonathan_TNL1 CGTGTTCCCGAAATGCTTCAGCTAAATCTCCGTTCTGATTCCTGACATGCGAAGGATCAACGTGATAGAATATTGGCAAA McIntosh_TNL1 CGTGTTTCTGAAATGCTTCGGCTAAATCTCCGTTCTGCTTCCTGATATGCGAAGGATCAACGTGATAGAATATTGGCAAA 330 340 350 360 370 380 390 400 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| 2249_TNL1 ACATG---CCCCAGTTTGCCTCTGCACTCCATGATCTTCACCAGCTCGTCAAGACACCAACTCGAATCCGCATACCTCCT Gala_TNL1 ACATGTCGCTCCAGTTTGTCTCTGCACTCCATGATCTTCACCAGCTCGTCAAGACACCAACTCGAATCCGCATACATCTT Braeburn_TNL1 ACATGTCGCCCCAGTTTGTCTCTGCACTCCATGATCTTCACCAGCTCGTCAAGACACCAACTCGAATCCGCATACATCTT Cox_TNL1 ACATGTCGCCCCAGTTTGTCTCTGCACTCCATGATCTTCACCAGCTCGTCAAGACACCAACTCGAATCCGCATACATCTT Delicious_TNL1 ACATGTCGCCCCAGTTTGTCTCTGCACTCCATGATCTTCACCAGCTCGTCAAGACACCAACTCGAATCCGCATACATCTT F2_TNL1 ACATGTCGCCCCAGTTTGTCTCTGCACTCCATGATCTTCACCAGCTCGTCAAGACACCAACTCGAATCCGCATACATCTT Golden_TNL1 ACATGTCGCTCCAGTTTGTCTCTGCACTCCATGATCTTCACCAGCTCGTCAAGACACCAACTCGAATCCGCATACATCTT Granny_TNL1 ACATGTCGCCCCAGTTTGTCTCTGCACTCCATGATCTTCACCAGCTCGTCAAGACACCAACTCGAATCCGCATACATCTT Jonathan_TNL1 ACATGTCGCCCCAGTTTGTCTCTGCACTCCATGATCTTCACCAGCTCGTCAAGACACCAACTCGAATCCGCATACATCTT McIntosh_TNL1 ACATGTCGCCCCAGTTTGTCTCTGCACTCCATGATCTTCACCAGCTCGTCAAGACACCAACTCGAATCCGCATACATCTT

Parents and 8 founders included

2nd choice SNPs

1st choice SNPs

FVbj3

Z13_SCAR CH02c06 SNP_region_20(8) B8

CH05e03

Rvi11

SNP_region_34(9)

CH03d01

0.1 0.1

2.0

0.7

0.7

0.8

3.8

Rvi11 Rvi11 (Vbj)

• Plant material: GD x A722-7, 145 plants

• 55 regions tested

• In 2 regions SNPs identified

• Vbj SNPs mapped between 0.7 and 3 cM

• 17 SNPs found in total

(Gygax et al., 2004)

Rvi2 (Vh2)

• Plant material: GD x X2250, 146 plants

• 44 regions tested

• In 8 regions SNPs identified

• Vh2 SNPs mapped between 0.7 and 2.8 cM

• 61 SNPS found in total

• Intense study and verification of the first mapping to define the correct map position

(Bus et al., 2005)

CH02b10CH2-Z13SCARSNP_region_53(8)SNP_region_41(1)CH05e03SNP_region_20.4(1)

CH-Vbj2SNP_region_21(15)SNP_region_23(5)

Rvi2

SNP_region_27(21)

L19SCARSNP_region_34.3(6)SNP_region_36(4)

CH03d01

0.7

0.7

0.7

0.7

1.4

Rvi2

Rvi4 (Vh4)

Hi22d06

SNP_region_TNL1(20)SNP_region_3(2)SNP_region_10(1)SNP_region_Vr2_3(3)Rvi4SNP_region_ARGH37(1)CH02c02a

Hi02a07SCAR-S22

2.1

0.4

1.3

Rvi4

• Plant material: Gala x X2249, 242 plants

• 30 regions tested

• In 5 regions SNPs identified

• Vh4 SNPs mapped at 0 cM

• 27 SNPs found in total

• Intense study and verification of the first mapping to define the correct map position

(Bus et al., 2005)

Rvi2 vs. Rvi11

CH02b10Z13_SCARSNP_region_53(8)SNP_region_41(1)CH05e03SNP_region_20(1)CH-Vbj2SNP_region_21(15)SNP_region_23(5)Rvi2

SNP_region_27(21)

L19SCARSNP_region_34(6)SNP_region_36(4)

CH03d01

0.7

0.7

0.7

0.7

1.4

Rvi2

Z13_SCARCH02c06SNP_region_20(8)B8

CH05e03

Rvi11

SNP_region_34(9)

FVbj3

CH03d01

0.10.1

2.0

0.7

0.7

0.8

3.8

Rvi11

• Indication that Rvi2 and Rvi11 may derive from the same region

• Allelic? • Phenotype of the two genes is

similar (SC vs. SN) • Difference due to difference level

of enhancement of the resistance cascade?

(Bus et al., 2011)

Rvi4 vs. Rvi15

• Indication that Rvi4 and Rvi15 may derive from the same region

• Allelic? • Phenotype of the two genes is the

same (PPP) • Rvi4 symptoms visible after 3-4

days, Rvi15 after 7 days • Difference due to difference level of

enhancement of the resistance cascade?

CH02c02a

S22_SCAR

Rvi4

1.0

4.0

Rvi4_Bus_et_2005

Hi22d06

SNP_region_TNL1(20)SNP_region_3(2)SNP_region_10(1)SNP_region_Vr2_3(3)Rvi4SNP_region_ARGH37(1)CH02c02a

Hi02a07S22_SCAR

2.1

0.4

1.3

Rvi4

CH02f06

ARGH37

Rvi15

ARGH17

CH02c02a

2.9

1.2

0.3

1.7

Rvi15

(Bus et al., 2011)

Conclusion

• SNP markers for resistance genes most used in breeding will be soon available.

• This will simplify marker assisted selection (MAS) and will allow a reduction of its costs .

• The precise mapping of the genes increases the precision of MAS and indicates the possible allelism of: – Rvi2 and Rvi11

– Rvi4 and Rvi15