East Malling Rootstock Breeding Club. NIAB EMR Update January 2018

Transcription

1 East Malling Rootstock Breeding Club NIAB EMR Update January 2018

2 Agenda Minutes and actions from previous meeting(s) Apple canker: PhD project (AG) & other projects (AK) EM trials completing in (AK) Pest and disease screening update (FF) Molecular Breeding update (FF) INN trial update (BE/CL) AOB

3 Actions from previous All comments/corrections to draft minutes of previous meeting to Mrs Chapple (by close of 22 September 2017) NIAB EMR team to undertake a survey to ascertain what markers and platforms had been published for rootstock work Ms Fernández - Data for 2017 harvest to be added and reviewed in January 2018; take decision whether to grub trial RF185 NIAB EMR team to update slides and report with 2017 data when available. Mr Essner to provide stool-bed information from PDV. Members of the group to discuss further at the January 2018 when all information is available and take decisions on selections to progress. Mr Essner to keep the Policy Group updated on progress on propagation of mother plants for PBR and commercialisation of AR Ms Fernández, Mrs Chapple to confirm date of next meeting (late January/early February 2018)

4 Agenda Minutes and actions from previous meeting(s) Apple canker: PhD project (AG) & other projects (AK) EM trials completing in (AK) Pest and disease screening update (FF) Molecular Breeding update (FF) INN trial update (BE/CL) AOB

5 The molecular basis of pathogenicity of Neonectria ditissima Antonio Gómez, Andrew D Armitage and Richard J Harrison

6 Neonectria ditissima Causative agent of the European apple canker Reported in almost all pome-producing regions of the world Wide host-range including apple, pear and broad-leaved trees Fungus attacks trees causing cankers and die back of young shoots, particularly damaging in young orchards Most modern apple varieties are susceptible and in extreme cases do not survive establishment in the orchard Fruit rot develops in the orchard or during storage

7 Life cycle

8 What is the problem? Huge economic losses - up to 10% of trees can be lost annually No effective control methods De-regulation of chemical fungicides Difficult to screen for the disease in breeding programmes N. ditissima is able to cross-infect a wide range of apple varieties differing in susceptibility to canker.

9 The molecular basis of pathogenicity There is no clear evidence of how N. ditissima penetrates into the host Necrotrophic lifestyle with a sophisticated mechanism of infection Cell wall degrading enzymes Proteins secreted from the pathogen that alter host processes The mechanism by which the pathogen is detected by the host it is still unknown

10 C141 PhD on the molecular basis of pathogenicity of N. ditissima Aims: Extend and deploy an accurate and quantifiable pathology test to measure quantitative differences in pathogenicity of differences N. ditissima isolates. Develop genomic resources and conduct a gene expression analysis in order to identify and characterise pathogenicity genes involved at different stages of the infection. Once candidates have been identified, validation of gene function by targeted disruption of the pathogen.

11 Host resistance responses Cut shoot test Leaf scar test Gomez-Cortecero, A. et al., 2016

12 N. ditissima pathogenicity test M9 potted trees were inoculated in a glasshouse with ten isolates of N. ditissima. Two axillary buds were inoculate. Buds cut off with a scalpel to simulate leaf petiole scar Lesion progression were measured at different time intervals.

13 AUDPC N. ditissima pathogenicity test More pathogenic Less pathogenic R37/15 R6/17-2 Ag02 R6/17-3 Ag06 R42/15 Isolate R41/15 R39/15 R45/15 Hg199 Area Under Disease Progress Curve. The AUDPC measures the disease throughout a period.

14 Sequencing N. ditissima Isolate accession CV Origin Year of isolation HG199 Gala Kent, UK 1999 Isolates of N. ditissima gathered worldwide will be sequenced by Illumina MiSeq. A reference genome will be generated using the innovative Long-read Pacbio sequencing technology. R09/05 Cox Kent, UK 2005 Ag02 Jazz Kent, UK 2016 Ag04 Conference Kent, UK 2015 Ag05 E Kent, UK 2016 Ag06 Unknown Kent, UK 2016 R6/17-2 Bramley Kent, UK 2017 R6/17-3 Bramley Kent. UK 2017 R37/15 Jonagold Belgium 1999 R39/15 Unknown Belgium 2006 R41/15 Wellant The Netherlands 2015 R42/15 Elstar The Netherlands 2015 R45/15 Elstar The Netherlands 2015 ND8 Royal Gala Brazil 2015 ND9 Royal Gala Brazil 2015

15 Sequencing strategy Raw Reads Genome Assembly Repeatmodeller, Repeatmasker & TransposonPSI RNAseq reads Repeatmasked assembly Braker1 prediction CodingQuarry prediction (pathogen mode) Predicted gene models

16 Genome architecture of N.ditissima Genome sequence of the R09/05 isolate Assembly statistics produced by Quast

17 Genome architecture of N.ditissima Predicted secreted effector proteins (Red) Assembly statistics produced by Quast

18 Genome architecture of N.ditissima Predicted Carbohydrate-active enzymes (Blue) Assembly statistics produced by Quast

19 Genome architecture of N.ditissima Hg199 R45/15 Reads aligning from other isolates Ag04 Assembly statistics produced by Quast

20 Genome statistics Organism Nd Nd Nd Nd Nd Nd Nd Nd Isolate R09/05 Hg199 R45/15 Ag04 Ag02 Ag05 ND8 R37/15 Origin UK UK The Netherlands UK UK UK Brazil Belgium A) Assembly stats: Total coverage (fold) Technology PacBio MiSeq MiSeq MiSeq MiSeq MiSeq MiSeq MiSeq B) Gene models: Assembly size (Mb) Contigs Largest contig (Kb) N50 (Kb) % Repeatmasked Total genes Braker transcripts CodingQuary transcripts Secreted Genes C) Effector candidates: Secreted & effector-like structure Secreted CAZYmes

21 Gene expression analysis RNA-Seq analysis to identify differentially expressed genes during the infection. Stem sections were collected from artificially inoculated apple plants. Differentially expressed genes will be identified in a susceptible and resistant cultivar. Golden Delicious M9 Reads will be mapped to the apple and to the N. ditissima reference genomes.

22 Future work Generate a high-quality genome sequence of N. ditissima. Comparative genomics of the isolates to identified genes hypothesised to be important in disease. RNA-Seq analysis to identify expressed effectors during infection. Functional confirmation of pathogenicity genes through targeted knockout.

23 Acknowledgments Dr. Andrew Armitage Dr. Helen Bates Dr. Rob Vickerstaff Dr. Robert Saville Dr. Richard Harrison Dr. Robert Jackson Thank you for your attention

24 Progress update BBSRC-LINK and AHDB projects on European apple canker East Malling Rootstock Club 31 st January 2018

25 The nature of resistance to Neonectria ditissima Repeating detached shoot screen 40 Malus species were screened with cut shoot test 15 rootstock genotypes (varieties and breeding lines) Propagation of biparental population based on results from detached shoot screens

26 Inoculated detached shoots Dormant shoots collected and inoculated in the glasshouse Three replicates per genotype Two lateral buds inoculated per shoot Conditions kept at C and a minimum humidity of 70% Volume and concentration of inoculum was 3 µl of 10 5 macroconidia/ml Single isolate of N.ditissima: Hg199 Lesion development will be measured with callipers at regular intervals

27 Treatments Treatment Rootstock Interstock Scion Number 1 M9 (EMLA) - Gala 2 M9 (337) - Gala 3 G.41 - Gala 4 G.11 - Gala 5 MM106 - Gala 6 M116 - Gala 7 M26 - Gala 8 M9 (337) Golden Delicious Gala TF223 - Improving integrated pest and disease management in tree fruit 9 AR295-6 (EMR-001) - Gala 10 AR440-1 (EMR-002) - Gala 11 AR486-1 (EMR-003) - Gala 12 AR628-2 (EMR-004) - Gala 13 AR680-2 (EMR-005) - Gala 14 AR (EMR-006) - Gala

28 Trial sites Site 1 East Egham Orchard, NIAB EMR, New Road, East Malling, Kent, ME19 6BJ 8 tree plots WINDBREAK M9 (337) interstock G.41 MM106 EMR-004 M9 (337) EMR-003 M116 EMR-002 GD EMR-005 M9 (EMLA) M116 EMR-006 M9 (EMLA) M116 EMR-001 G.11 Grid reference , Planted 29 th March 2017 The site is situated amongst mature orchards in which Neonectria Description of planting site: dittisima inoculum is prevalent providing opportunities for natural infection. Trial layout: M116 EMR-004 G.41 M9 (EMLA) EMR-002 EMR-004 M9 (337) M9 (EMLA) M9 (337) M9 (337) EMR-003 interstock EMR-002 EMR-005 EMR-006 EMR-005 MM106 interstock GD GD EMR-002 M9 (337) MM106 EMR-001 M9 (337) MM106 EMR-005 EMR-003 M26 EMR-001 EMR-003 M26 G.41 G.11 EMR-006 M26 EMR-006 G.11 M9 (337) interstock G.11 EMR-001 M26 G.41 EMR-004 GD 4 replicates of 8 tree plots, arranged over four blocks (as determined by colour) = 4 spare tree stations ALLEY WAY N WOODLAND Site 2 Herridges Orchard, Ketford Road, Poolhill nr. Newent, Gloucestershire. GL18 1LW 6 MORE ROWS OF APPLE TREES Row 1 5G 5 2G 2 2G 13 2G 8 2G 11 2G 9 10G Row 2 9G 4 2G 2G 12 2G 10 2G 14 2G 3 6G Grid reference , Planted 15 th March 2017 Row 3 5G 1 2G 6 2G 7 2G 11 2G 1 2G 7 9G Row 4 2G 2G 14 2G 12 2G 4 2G 8 2G 9 10G The trial was planted on the site of an old Cox orchard. 2 cox Row 5 5G 6 2G 3 2G 13 2G 2 2G 5 2G 10 6G Description of planting site: trees were left in the ground between each plot to serve as an inoculum source throughout the trial. Row 6 8G 14 2G 5 2G 2G 7 2G 9 2G 13 2G Row 7 2G 3 2G 8 2G 4 2G 6 2G 1 2G 10 8G Trial layout: Row 8 6G 2 2G 11 2G 12 2G 10 2G 5 2G 13 2G Row 9 2G 3 2G 11 2G 6 2G 12 2G 9 2G 1 6G 4 replicates of 10 tree plots per treatment. Each plot separated by mature Cox trees Row 10 2G 7 2G 4 2G 8 2G 2G 14 2G 2 2G 4 MORE ROWS OF APPLE TREES HEDGE

29 Assessments Natural infection Assessed throughout the project (3 more years) Very low levels in Autumn 2017 assessment Autumn infection window will lead to more cankers come Spring 18 Artificial infection Leaf and bud scars inoculated in Autumn 17 Incidence and severity assessed in Spring 18

30 Agenda Minutes and actions from previous meeting(s) Apple canker EM trials completing in (AK) Pest and disease screening update (FF) Molecular Breeding update (FF) INN trial update (BE/CL) AOB

31 On going trials - Apple Second stage: EE207 (planted in 2010) VF224 (planted in 2010) SC250 (planted in 2014) Eufrin trial (planted in 2017) First stage: RF185 (planted 2012 but not promising) The data presented is not final vigour evaluation for VF224 is still ongoing as is for RF185: As soon as data analysis is completed, we will circulate our recommendations and ask for feedback to finalise decisions

32 Second stage apple trial (EE207) Planted 2010 Five NIAB-EMR rootstock selections : AR852-3 AR839-9 B24 R104 R59 Standards M27, M9 and M26 Royal Gala & Braeburn under conventional orchard management Statistical analysis performed with Anova in R

33 EE207 Tree size: Girth a a ab abc a abc a abc abc c abc abc bc c c c The p-value for rootstock effect was <0.001 Significant interaction rootstock:scion (p=0.046)

34 EE207 Tree volume 2016

35 EE207 Suckering a a a ab b b b b Significant effect of roostock (p<0.001) No interaction scion:rootstock

36 EE207 Fruit yield 2017 a a ab ab abc abc bc c c ab ab ab abc bc bc c The p-value for rootstock effect was <0.001 for both fruit weight and number No significant interaction between rootstock and scion

37 EE207

38 EE207 No significant effect of rootstock (p=0.09) No significant interaction between scion and rootstock

39 EE207 Cumulative yield

40 EE207 No significant effect of rootstock on bitter pit Significant effect of scion (p=0.01)

41 Second stage apple trial (VF224) Planted 2010 Four NIAB-EMR rootstock selections : AR AR809-3 AR R80 Standards M116 & M106 Red Falstaff under organic orchard management

42 VF224 - Vigour a a a ab ab b a a a a ab b Significant effect of rootstock on both tree volume and girth (p<0.001)

43 VF224 Suckering

44 VF224 Red Falstaff: Fruit yield 2017 a ab ab ab ab b a ab ab ab ab b Significant effect of rootstock on total fruit yield and total number of fruits per tree(p<0.05)

45 VF224 Total yield by fruit size

46 VF224 Bitter pit No significant effect of rootstock on incidence of bitter pit

47 VF224 Yield efficiency No significant effect of rootstock on either measure of yield efficiency

48 VF224 Cumulative yield

49 INN AR M.27 x M.M.106 < M.9 < M.9 Re-test 2018 High INN AR M.27 x M.M.106 > M.116 > M.116 Susceptible High Fully susceptible Susceptible? AR69-7 AR op Susceptible - V. susceptible (field) Very poor De-selected - AR M.M.106 x M.27 ~ M.116 ~ M.116 Resistant? Intermediate-Susceptible INN AR M.27 x M.M.106 ~ Cepiland > Cepiland Susceptible Very high - AR M.9 x M.27 Susceptible Intermediate-Susceptible INN AR295-6 R.5 x Ottawa 3 ~ > M.9 Similar / > M.9 Susceptible Very low AR440-1 M.25 x M.27 Susceptible? High INN AR486-1 Ottawa 3 x M.7 Susceptible Medium Intermediate-Susceptible AR628-2 Ottawa 3 x M.M.106 Test 2018 High Fully susceptible INN AR680-2 M.26 x M.27 ~10% < Cepiland > Size & > Yield Susceptible High Fully susceptible AR682-6 M.26 x MI.793 Resistant Medium/High? AR M.26 x M.9 Susceptible? Medium/High? Fully susceptible VF224 AR809-3 R.80 x M.26 Susceptible High Inconclusive VF224/INN AR MI.793 x M.9 ~ 25% > Cepiland ~ 25% > Cepiland Test 2018 Low INN AR M.3 x M.1 ~ Cepiland ~ Cepiland Susceptible Low/Medium? EE207/INN AR839-9 M.7 x M.27 ~ 50% > Cepiland Good Yield & Size Susceptible Low EE207 AR852-3 AR o.p. Test 2018 High? Intermediate-Susceptible EE207 B24 AR x AR Resistant Medium/High? Intermediate-Tolerant Inconclusive EE207 R104 AR x AR Resistant High? Fully susceptible Resistant? EE207 R59 AR x AR Re-test 2018 High? VF224 R80 (HDC)? Re-test 2018 Medium/High? VF224 M.116 M.M.106 x M.27 Resistant (High) Fully susceptible VF224 M.M.106 Northern Spy x M.1 Resistant (High) EE207 M.9 - Susceptible Susceptible Fully susceptible EE207 M.26 EE207 M.27 M.9 x M.13 Proposed selection/deselection table Trial (s) Genotype Parentage Vigour Productivity WAA response FB Susceptibility Canker* P. cactorum Propagation Decision

50 Agenda Minutes and actions from previous meeting(s) Apple canker EM trials completing in (AK) Pest and disease screening update (FF) Molecular Breeding update (FF) INN trial update (BE/CL) AOB

51 Pest and Disease screening overview Fireblight: Apple: No further testing at Agroscope is possible for Pear: Some results expected Spring 2018 (INRA) WAA: OK season in 2017 for seedlings HWC were poor so not many selections (re-test 2018) P cactorum: HWC were poor no conclusive infection 2018: - cut shoot test - seedling test (pathogenicity) - HWC

52 Genotype Parentage WAA response FB Susceptibility Canker* P. cactorum AR M.27 x M.M.106 Re-test 2018 High AR M.27 x M.M.106 Susceptible High Fully susceptible Susceptible? AR69-7 AR op Susceptible - V. susceptible (field) AR M.M.106 x M.27 Resistant? Intermediate-Susceptible AR M.27 x M.M.106 Susceptible Very high AR M.9 x M.27 Susceptible Intermediate-Susceptible AR295-6 R.5 x Ottawa 3 Susceptible Very low AR440-1 M.25 x M.27 Susceptible? High AR486-1 Ottawa 3 x M.7 Susceptible Medium Intermediate-Susceptible AR628-2 Ottawa 3 x M.M.106 Test 2018 High Fully susceptible AR680-2 M.26 x M.27 Susceptible High Fully susceptible AR682-6 M.26 x MI.793 Resistant Medium/High? AR M.26 x M.9 Susceptible? Medium/High? Fully susceptible AR809-3 R.80 x M.26 Susceptible High Inconclusive AR MI.793 x M.9 Test 2018 Low AR M.3 x M.1 Susceptible Low/Medium? AR839-9 M.7 x M.27 Susceptible Low AR852-3 AR o.p. Test 2018 High? Intermediate-Susceptible B24 AR x AR Resistant Medium/High? Intermediate-Tolerant Inconclusive R104 AR x AR Resistant High? Fully susceptible Resistant? R59 AR x AR Re-test 2018 High? * GH cut-shoot test 2017 only, unless indicated GH inoculation of harwood cuttings 2016 (Responses in brackets) are those reported for the genotype but have not been re-tested by NIAB EMR

53 WAA Observed Expected (1 gene) Expected (2 gene) Expected (3 gene) Total R S R S R S R S M606 Evereste (S) x C.G.202 ( R) Plan for 2018: Markers linked to WAA resistance will be tested in part of this population clarification of inheritance & marker validation FB markers will be tested in the R seedlings: Seedlings with both resistances will be bench grafted alongside controls for agronomic evaluation (cost analysis)

54 Agenda Minutes and actions from previous meeting(s) Apple canker EM trials completing in (AK) Pest and disease screening update (FF) Molecular Breeding update (FF) INN trial update (BE/CL) AOB

55 Molecular Breeding Review of all usable markers on-going: Update of K Evans s 2010 review paper plus small scale validation on our breeding material currently being carried out by our new team member (Suzanne Litthauer) FB_R5 markers: FB_E markers: ongoing new unpublished info for Agroscope (2018) Dwarfing markers: ongoing need to compare Rb1-3 with Dw1&2 WAA markers:

56 Genotypes tested FB_R5 Full resistance is identified Low susceptibility is not linked to the R5_locus markers Going forward we will use FEM47 & FEM19 only Fb_Mr5 Marker FEM14 FEM47 FEM19 Position cm cm cm Bud /158 Evereste 183/ /148 CG11 181/ / /159 CG30 181/ / /157 CG41 181/ / /163 CG / / /163 M /163 M /187 (194) 159 M / /163 M.M / M. floribunda_ / /159 MIS 181/ /148 Robusta 5 181/ / /150 Torstein /172 AR /164 M /164 AR /163 AR /187 (193) 159 AR /172 AR / AR / /163 AR / AR / /163 AR /171 AR / AR (182) 159 M306_ /172 M306_ /172 M306_ /157 M306_ /172

57 Genotypes tested FB_E: ChFbE08 problematic for R5 germplas Need to add ChFbE06 & ChFbE09 for accuracy (Patocchi p. comm) Fb_Ev Marker ChFbE01 ChFbE02 ChFbE06 ChFbE08 ChFbE09 Position cm Fb_E (-4Kb) Fb_E (+10Kb) cm cm Bud / /248 Evereste 241/ /248 CG /248 CG /248 CG /248 CG /248 M / /248 M / /246 M / /246 M.M /246 M. floribunda_ / MIS 241/ / Robusta /248 Torstein /248 AR M AR / /148 AR / AR AR /246 AR / /248 AR /248 AR /248 AR / /246 AR /246 AR / /246 M306_ / /241 M306_ / /241 M306_ /248 M306_ / /248

58 Agenda Minutes and actions from previous meeting(s) Apple canker EM trials completing in (AK) Pest and disease screening update (FF) Molecular Breeding update (FF) INN trial update (BE/CL) AOB