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

Actions from previous • 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)

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 of 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 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.

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 1800 More 1600 pathogenic 1400 1200 AUDPC 1000 800 600 400 Less 200 pathogenic 0 5 2 2 3 6 5 5 5 5 9 1 - 0 - 0 1 1 1 1 9 7 7 / g g / / / / 1 7 1 1 2 1 9 5 A A g 3 / / 4 4 3 4 6 6 H R R R R R R R Isolate Area Under Disease Progress Curve. The AUDPC measures the disease throughout a period.

Isolate Sequencing N. ditissima CV Origin Year of isolation accession HG199 Gala Kent, UK 1999 R09/05 Cox Kent, UK 2005 • Isolates of N. ditissima Ag02 Jazz Kent, UK 2016 gathered worldwide will be Ag04 Conference Kent, UK 2015 Ag05 E830-102 Kent, UK 2016 sequenced by Illumina MiSeq. Ag06 Unknown Kent, UK 2016 R6/17-2 Bramley Kent, UK 2017 • A reference genome will be R6/17-3 Bramley Kent. UK 2017 generated using the innovative R37/15 Jonagold Belgium 1999 Long-read Pacbio sequencing R39/15 Unknown Belgium 2006 R41/15 Wellant The Netherlands 2015 technology. R42/15 Elstar The Netherlands 2015 R45/15 Elstar The Netherlands 2015 ND8 Royal Gala Brazil 2015 ND9 Royal Gala Brazil 2015

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

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 Hg199 R45/15 Ag04 Reads aligning from other isolates Assembly statistics produced by Quast

Genome statistics Organism Nd Nd Nd Nd Nd Nd Nd Nd Isolate R09/05 Hg199 R45/15 Ag04 Ag02 Ag05 ND8 R37/15 The Origin UK UK UK UK UK Brazil Belgium Netherlands 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 • 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 Golden Delicious M9 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 31 st 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 10-20 0 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

Treatments Treatment Rootstock Interstock Scion Number 1 M9 (EMLA) - Gala TF223 - Improving integrated pest 2 M9 (337) - Gala and disease management in tree fruit 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