East Malling Rootstock Breeding Club NIAB EMR Update January 2018 - - PowerPoint PPT Presentation

East Malling Rootstock Breeding Club NIAB EMR Update – January 2018

Agenda

• Minutes and actions from previous meeting(s)
• Apple canker: PhD project (AG) & other projects (AK)
• EM trials completing in 2017-18 (AK)
• Pest and disease screening update (FF)
• Molecular Breeding update (FF)
• INN trial update (BE/CL)
• AOB

• All – email 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 2016-17 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 295-6

• Ms Fernández, Mrs Chapple to confirm date of next meeting (late January/early February 2018)

Actions from previous

Agenda

• Minutes and actions from previous meeting(s)
• Apple canker: PhD project (AG) & other projects (AK)
• EM trials completing in 2017-18 (AK)
• Pest and disease screening update (FF)
• Molecular Breeding update (FF)
• INN trial update (BE/CL)
• AOB

The molecular basis of pathogenicity

• f Neonectria ditissima

Antonio Gómez, Andrew D Armitage and Richard J Harrison

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

Life cycle

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.

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

C141 – PhD on the molecular basis of pathogenicity of N. ditissima

• 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.

Aims:

Host resistance responses

Leaf scar test Cut shoot test

Gomez-Cortecero, A. et al., 2016

• 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.

• N. ditissima pathogenicity test

200 400 600 800 1000 1200 1400 1600 1800 R 3 7 / 1 5 R 6 / 1 7

• 2

A g 2 R 6 / 1 7

• 3

A g 6 R 4 2 / 1 5 R 4 1 / 1 5 R 3 9 / 1 5 R 4 5 / 1 5 H g 1 9 9

Isolate AUDPC

Less pathogenic More pathogenic

Area Under Disease Progress Curve. The AUDPC measures the disease throughout a period.

Sequencing N. ditissima

• Isolates
• f

N. ditissima gathered worldwide will be sequenced by Illumina MiSeq.

• A reference genome will be

generated using the innovative Long-read Pacbio sequencing technology.

Isolate accession CV Origin Year of isolation HG199 Gala Kent, UK 1999 R09/05 Cox Kent, UK 2005 Ag02 Jazz Kent, UK 2016 Ag04 Conference Kent, UK 2015 Ag05 E830-102 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

Sequencing strategy

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

Genome architecture of N.ditissima

Genome sequence of the R09/05 isolate

Assembly statistics produced by Quast

Genome architecture of N.ditissima

Predicted secreted effector proteins (Red)

Assembly statistics produced by Quast

Genome architecture of N.ditissima

Predicted Carbohydrate-active enzymes (Blue)

Assembly statistics produced by Quast

Genome architecture of N.ditissima

Reads aligning from

• ther isolates

Assembly statistics produced by Quast

Hg199 R45/15 Ag04

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)

• 74

86 30 96 77 159 76 Technology PacBio MiSeq MiSeq MiSeq MiSeq MiSeq MiSeq MiSeq Assembly size (Mb) 45.9 45.4 45.2 46.4 44.8 45.06 45.86 44.67 Contigs 70 849 969 728 779 800 613 916 Largest contig (Kb) 4130 614 704 643 572 619 727 551 N50 (Kb) 1241 182 170 160 152 156 192 144 % Repeatmasked 12.43 10.88 10.73 12.85 10.11 10.41 11.96 9.19 B) Gene models: Total genes 13746 13723 13624 13659 14042 14027 14002 14020 Braker transcripts 12848 12818 12635 12805 13282 13269 13257 13283 CodingQuary transcripts 898 905 989 854 761 758 745 737 Secreted Genes 1017 1009 1005 1006 992 966 951 989 C) Effector candidates: Secreted & effector-like structure 170 165 167 166 160 155 164 163 Secreted CAZYmes 286 285 287 283 281 279 275 272

Gene expression analysis

Golden Delicious M9

• 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.

• Reads will be mapped to the apple and to the
• N. ditissima reference genomes.

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.

Acknowledgments

• Dr. Andrew Armitage
• Dr. Helen Bates
• Dr. Rob Vickerstaff
• Dr. Robert Saville
• Dr. Richard Harrison
• Dr. Robert Jackson

Thank you for your attention

Progress update

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

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

Inoculated detached shoots

• Dormant shoots collected and inoculated in the

glasshouse

• Three replicates per genotype

– Two lateral buds inoculated per shoot

• Conditions kept at 10-200C and a minimum humidity
• f 70%
• Volume and concentration of inoculum was 3 µl of 105

macroconidia/ml

• Single isolate of N.ditissima: Hg199
• Lesion development will be measured with callipers at

regular intervals

Treatment Number Rootstock Interstock Scion 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 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 AR86-1-20 (EMR-006)

• Gala

Treatments

TF223 - Improving integrated pest and disease management in tree fruit

Site 2 Herridges Orchard, Ketford Road, Poolhill nr. Newent,

• Gloucestershire. GL18 1LW

Grid reference 51.966956, -2.3953805 Planted 15th March 2017 Description of planting site: The trial was planted on the site of an old Cox orchard. 2 cox trees were left in the ground between each plot to serve as an inoculum source throughout the trial. Trial layout: 4 replicates of 10 tree plots per treatment. Each plot separated by mature Cox trees

Site 1 East Egham Orchard , NIAB EMR, New Road, East Malling, Kent, ME19 6BJ Grid reference 51.287861, 0.43831340 Planted 29th March 2017 Description of planting site: The site is situated amongst mature orchards in which Neonectria dittisima inoculum is prevalent providing opportunities for natural infection. Trial layout: 4 replicates of 8 tree plots, arranged over four blocks (as determined by colour)

Trial sites

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

Agenda

• Minutes and actions from previous meeting(s)
• Apple canker
• EM trials completing in 2017-18 (AK)
• Pest and disease screening update (FF)
• Molecular Breeding update (FF)
• INN trial update (BE/CL)
• AOB

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 e-mail feedback to finalise decisions

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

EE207 Tree size: Girth

a a a a ab abc abc abc abc abc c abc bc c c c

The p-value for rootstock effect was <0.001 Significant interaction rootstock:scion (p=0.046)

EE207 Tree volume 2016

EE207 Suckering

Significant effect of roostock (p<0.001) No interaction scion:rootstock

a a a ab b b b b

EE207 – Fruit yield 2017

a ab c c bc abc abc ab

The p-value for rootstock effect was <0.001 for both fruit weight and number No significant interaction between rootstock and scion

a ab bc c bc abc ab ab

EE207

EE207

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

EE207 Cumulative yield 2011-2017

EE207

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

Second stage apple trial (VF224)

• Planted 2010
• Four NIAB-EMR rootstock selections :

AR10-3-9 AR809-3 AR835-11 R80

• Standards M116 & M106
• ‘Red Falstaff’ under organic orchard management

VF224 - Vigour

Significant effect of rootstock on both tree volume and girth (p<0.001)

a a a a ab b a a a ab ab b

VF224 Suckering

VF224 Red Falstaff: Fruit yield 2017

Significant effect of rootstock on total fruit yield and total number of fruits per tree(p<0.05)

a ab ab ab ab b a ab ab ab ab b

VF224 Total yield by fruit size

VF224 Bitter pit

No significant effect of rootstock on incidence of bitter pit

VF224 Yield efficiency

No significant effect of rootstock on either measure of yield efficiency

VF224 Cumulative yield 2011-2017

Proposed selection/deselection table

Trial (s) Genotype Parentage Vigour Productivity WAA response FB Susceptibility Canker*

• P. cactorum† Propagation

Decision INN AR10-2-5 M.27 x M.M.106 < M.9 < M.9 Re-test 2018 High INN AR10-3-9 M.27 x M.M.106 > M.116 > M.116 Susceptible High Fully susceptible Susceptible? AR69-7 AR10-2-6 op Susceptible

• V. susceptible (field)

Very poor De-selected

• AR86-1-20

M.M.106 x M.27 ~ M.116 ~ M.116 Resistant? Intermediate-Susceptible INN AR120-242 M.27 x M.M.106 ~ Cepiland > Cepiland Susceptible Very high

• AR360-19

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? - 2018 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? AR801-11 M.26 x M.9 Susceptible? - 2018 Medium/High? Fully susceptible VF224 AR809-3 R.80 x M.26 Susceptible High Inconclusive VF224/INN AR835-11 MI.793 x M.9 ~ 25% > Cepiland ~ 25% > Cepiland Test 2018 Low INN AR837-19 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 AR362-16 o.p. Test 2018 High? Intermediate-Susceptible EE207 B24 AR10-2-5 x AR86-1-22 Resistant Medium/High? Intermediate-Tolerant Inconclusive EE207 R104 AR134-31 x AR86-1-22 Resistant High? Fully susceptible Resistant? EE207 R59 AR134-31 x AR86-1-22 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

Agenda

• Minutes and actions from previous meeting(s)
• Apple canker
• EM trials completing in 2017-18 (AK)
• Pest and disease screening update (FF)
• Molecular Breeding update (FF)
• INN trial update (BE/CL)
• AOB

• Fireblight:

– Apple: No further testing at Agroscope is possible for 2018-19 – 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

Pest and Disease screening – overview

Genotype Parentage WAA response FB Susceptibility Canker*

• P. cactorum†

AR10-2-5 M.27 x M.M.106 Re-test 2018 High AR10-3-9 M.27 x M.M.106 Susceptible High Fully susceptible Susceptible? AR69-7 AR10-2-6 op Susceptible

• V. susceptible (field)

AR86-1-20 M.M.106 x M.27 Resistant? Intermediate-Susceptible AR120-242 M.27 x M.M.106 Susceptible Very high AR360-19 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? - 2018 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? AR801-11 M.26 x M.9 Susceptible? - 2018 Medium/High? Fully susceptible AR809-3 R.80 x M.26 Susceptible High Inconclusive AR835-11 MI.793 x M.9 Test 2018 Low AR837-19 M.3 x M.1 Susceptible Low/Medium? AR839-9 M.7 x M.27 Susceptible Low AR852-3 AR362-16 o.p. Test 2018 High? Intermediate-Susceptible B24 AR10-2-5 x AR86-1-22 Resistant Medium/High? Intermediate-Tolerant Inconclusive R104 AR134-31 x AR86-1-22 Resistant High? Fully susceptible Resistant? R59 AR134-31 x AR86-1-22 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

WAA

Total R S R S R S R S M606 Evereste (S) x C.G.202 ( R) 280 42 238 140 140 70 210 35 245 Observed Expected (1 gene) Expected (2 gene) Expected (3 gene)

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)

Agenda

• Minutes and actions from previous meeting(s)
• Apple canker
• EM trials completing in 2017-18 (AK)
• Pest and disease screening update (FF)
• Molecular Breeding update (FF)
• INN trial update (BE/CL)
• AOB

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)

Molecular Breeding

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: 2018-2019

• Full resistance is identified
• Low susceptibility is not linked

to the R5_locus markers

• Going forward we will use

FEM47 & FEM19 only

FB_R5

Marker FEM14 FEM47 FEM19 Position

• 0.07 cM
• 0.07 cM

+0.31 cM Bud9 187 215 130/158 Evereste 183/189 215 138/148 CG11 181/189 209/215 150/159 CG30 181/187 209/215 150/157 CG41 181/187 209/215 150/163 CG202 181/187 209/215 150/163 M.9 187 215 157/163 M.13 181/187 (194) 159 M.27 181/187 215 159/163 M.M.106 187/189 215 157

• M. floribunda_821

181/183 209 148/159 MIS 181/189 215 136/148 Robusta 5 181/187 209/215 134/150 Torstein 187 215 136/172 AR86-1-20 187 215 157/164 M.116 187 215 157/164 AR295-6 187 215 133/163 AR440-1 181/187 (193) 159 AR680-2 187 215 163/172 AR682-6 187/189 215 157 AR801-11 187/189 215 159/163 AR809-3 187/189 215 159 AR835-11 181/187 215 159/163 AR837-19 187 215 163/171 AR839-9 187/189 215 157 AR852-3 181 (182) 159 M306_006 187 215 157/172 M306_020 187 215 163/172 M306_079 187 215 134/157 M306_189 187 215 163/172 Fb_Mr5

Genotypes tested

FB_E:

Marker ChFbE01 ChFbE02 ChFbE06 ChFbE08 ChFbE09 Position

• 0.11 cM

Fb_E (-4Kb) Fb_E (+10Kb) +0.07 cM +0.19 cM Bud9 241 239/248 241/248 Evereste 241/247 231 241/248 CG11 240 237 239/248 CG30 240 237 239/248 CG41 240 237 239/248 CG202 240 248 239/248 M.9 240 241/248 241/248 M.13 240 238/246 239/246 M.27 240 241/246 241/246 M.M.106 240 241 241/246

• M. floribunda_821

242 231/246 248 MIS 241/261 231/240 239 Robusta 5 240 237 237/248 Torstein 240 227 226/248 AR86-1-20 242 241 241 M.116 242 241 239 AR295-6 240 241/248 241/148 AR440-1 242 239/241 241 AR680-2 242 241 241 AR682-6 240 241 241/246 AR801-11 240 241/248 241/248 AR809-3 240 248 239/248 AR835-11 241 246 239/248 AR837-19 240 241/246 240/246 AR839-9 240 241 241/246 AR852-3 241 241/246 241/246 M306_006 241 238/241 238/241 M306_020 242 238/241 238/241 M306_079 241 241 240/248 M306_189 242 241/248 240/248

Genotypes tested

Fb_Ev

• ChFbE08 – problematic for R5

germplas

• Need to add ChFbE06 & ChFbE09

for accuracy (Patocchi p. comm)

Agenda

• Minutes and actions from previous meeting(s)
• Apple canker
• EM trials completing in 2017-18 (AK)
• Pest and disease screening update (FF)
• Molecular Breeding update (FF)
• INN trial update (BE/CL)
• AOB