IV INTERNATIONAL WORKSHOP ON HOST-PARASITE INTERACTIONS IN FORESTRY

HighHigh ResolutionResolution GeneticGenetic andand PhysicalPhysical MappingMapping ofof EasternEastern FilbertFilbert BlightBlight ResistanceResistance inin HazelnutHazelnut

Vidyasagar R. Sathuvalli and Shawn A. Mehlenbacher Department of Horticulture, Oregon State University EasternEastern FilbertFilbert BlightBlight inin HazelnutHazelnut

¾Major devastating disease ¾First identified in southwest Washington – 1968 ¾First discovered in Willamette valley – 1986 ¾Moved Southward @ 2‐3 km per year ¾More than 60 % of Oregon’s hazelnut orchards are affected or in close proximity to diseased orchards. EasternEastern FilbertFilbert BlightBlight inin HazelnutHazelnut

¾ Causal fungus: Anisogramma anomala ¾ Division: Ascomycota ¾ Sub‐division: Pezizomycotina

extension.oregonstate.edu/.../EC149902‐02.jpg ¾ Class: Sordariomycetes ¾ Order: Diaporthales ¾ Family: Valsaceae (Dict. Fung., 2001)

Jose R. Liberato DPI&F Jose R. Liberato DPI&F Jose R. Liberato DPI&F DistributionDistribution ofof AnisogrammaAnisogramma anomalaanomala

2004‐05

Adapted & Modified from http://www.eppo.org

Map by Jay W. Pscheidt and Pat Grimaldi, 2006 LifeLife CycleCycle ofof AnisogrammaAnisogramma anomalaanomala

Wet conditions & Budbreak

13‐16 months

Chilling & dormancy DiseaseDisease ResistanceResistance BreedingBreeding ¾EFB resistance –Important objective of hazelnut breeding program at OSU ¾‘Gasaway’ resistance –mostly employed ¾Resistance controlled by dominant allele at single locus ¾Most advanced selections –‘Gasaway’ resistance ¾‘Jefferson’ recently released EFB resistance cultivar

Barcelona Jefferson Susceptible Resistant

Gasaway Jefferson OutlineOutline ofof mapmap‐‐basedbased cloningcloning approachapproach

Disease resistance Linked Markers

Mapping BAC end markers

Krattinger et al., 2009 Isolate disease resistance gene RequirementsRequirements forfor mapmap‐‐basedbased cloningcloning approachapproach

¾ A genetic map ¾ A large mapping population and Identification of recombinants ¾ A Bacterial Artificial Chromosome (BAC) library ¾ Probes for BAC library screening for chromosome walking GeneticGenetic linkagelinkage mapmap ofof hazelnuthazelnut

¾ Mehlenbacher et al. (2006) constructed a genetic linkage map for hazelnut with RAPD and SSR markers ¾ ‘Gasaway’ resistance in linkage group 6 ¾ Nine RAPD markers within 7.5cM of resistance RequirementsRequirements forfor mapmap‐‐basedbased cloningcloning approachapproach

¾ A genetic map ¾ A large mapping population and Identification of recombinants ¾ A Bacterial Artificial Chromosome (BAC) library ¾ Probes for BAC library screening for chromosome walking MappingMapping PopulationPopulation ¾ In 2007, controlled crosses between OSU 252.146 and OSU 414.062 generated 1488 seedlings ƒ 07001 – 1080 seedlings ƒ 07002 – 408 seedlings

07002 is from reciprocal cross, OSU 414.062 x OSU 252.146 FineFine scalescale geneticgenetic mappingmapping Marker1 0.0 152-800 ¾ DNA was extracted from 1488 seedlings in 2008 ¾ Screened for the presence of RAPD markers 152‐800 and 268‐580 1.4 R-locus ¾ Both markers present – Assumed to be resistant ¾ Both markers absent – Assumed to be susceptible ¾ One marker present and the other absent – inoculated in the greenhouse ¾ Recombinant seedlings were used to map new BAC end markers 4.4 268-580 Marker2 RAPDRAPD MarkerMarker ScreeningScreening

UBC152‐800

______+ _ + _ + +

UBC268‐580

______+ + + +

Inoculated in the greenhouse for disease response DiseaseDisease InoculationsInoculations Carried out in locked greenhouse 1 2

13‐16 months later

3 4 HighHigh resolutionresolution mappingmapping ofof EFBEFB resistanceresistance regionregion withwith RAPDRAPD markersmarkers

¾ Screening of 1488 seedlings identified 87 recombinants between RAPD markers 152‐ 800 and 268‐580. ¾ Linkage map was constructed with 7 RAPDs, 2 HRM and 1 SCAR markers using JoinMap v.4.0 ¾ Resistance is flanked by two RAPD markers W07‐375 and X01‐825 at 0.05 and 0.06 cM, respectively. RequirementsRequirements forfor mapmap‐‐basedbased cloningcloning approachapproach

¾ A genetic map ¾ A large mapping population and Identification of recombinants ¾ A Bacterial Artificial Chromosome (BAC) library ¾ Probes for BAC library screening for chromosome walking BACBAC librarylibrary ofof HazelnutHazelnut

¾ Constructed for ‘Jefferson’ ¾ Cloning enzyme ‐ MboI ¾ Vector ‐ pECBAC1 (BamHI site) ¾ Average insert size – 117kb ¾ Genome coverage – 12x ¾ 39,936 clones arrayed in 104 384‐well plates RequirementsRequirements forfor mapmap‐‐basedbased cloningcloning approachapproach

¾ A genetic map ¾ A large mapping population and Identification of recombinants ¾ A Bacterial Artificial Chromosome (BAC) library ¾ Probes for BAC library screening for chromosome walking ProbesProbes forfor ScreeningScreening BACBAC librarylibrary

¾ 9 RAPD markers close to resistance where cloned and sequenced ¾ Two sequence characterized amplified region (SCAR) markers were designed ¾ These new SCAR markers were used to screen the BAC library

152‐800 268‐580 ScreeningScreening ofof BACBAC librarylibrary Pooling and Screening by PCR

Plate pools - 104 Row pools - 16 per plate Extract DNA

Column pools - 24 per plate

18 SCARs from Screen Plate pools 9 RAPD markers Screening Screen Row and Column pools twice helps 17 avoid false

Row pool E positives and Identify the positive clones other PCR artifacts

Column Sequence the BAC ends Hit:78‐Ex17 pool ChromosomeChromosome WalkingWalking

Sequence the BAC ends

Design new primers from BAC ends

Map the new markers

Screen the BAC library with new BAC end probes

Identify new BACs

Identify the BACs carrying resistance MarkersMarkers fromfrom BACBAC endend sequencessequences

¾ Lack of polymorphism in the BAC end markers –a major constraint ¾ Various kinds of markers were developed from BAC end sequences ƒ Sequence Characterized Amplified Region (SCAR) ƒ Single Stranded Conformational Polymorphism (SSCP) ƒ Simple Sequence Repeat (SSR) ƒ High Resolution Melting (HRM) ƒ Cleaved Amplified Polymorphic Sequence (CAPS) ¾ Primers were designed either using Primer 3 or LightScanner primer design (HRM) Software ChromosomeChromosome WalkingWalking

¾ Initial Screening with SCARs developed from RAPD markers identified 36 BACs ¾ Two further rounds of chromosome walking were carried out from BAC end markers ¾ A total of 93 BACs were identified FineFine mappingmapping ofof thethe EFBEFB resistanceresistance regionregion BACBAC endend SCARSCAR markersmarkers

RPSPRSRS SSSS RPSPRSRS SSSS

BE01 BE33 2% Agarose –90v‐6hrs 2% Agarose –90v‐2.5hrs BACBAC endend SSCPSSCP MarkersMarkers

RPSP RSRS SS SS

0.5X MDE® Gel* ‐ 4.0 Watt –12hrs

BE04 - SSCP RPSP RSRS SS SS

0.5X MDE® Gel* ‐ 4.0 Watt –18hrs

BE05 ‐ SSCP * Lonza Rockland, Inc., Rockland, ME, USA

BACBAC endend HRMHRM markersmarkers

TBE126 TBE_II_14

Susceptible melt curve Resistant melt curve Susceptible melt curve HRMHRM‐‐ MarkersMarkers fromfrom RAPDsRAPDs

HRM‐based RAPD marker X01‐825 HRM‐based RAPD marker W07‐375

Susceptible melt curve Resistance melt Susceptible melt curve curve

FineFine mappingmapping ofof thethe EFBEFB resistanceresistance regionregion

¾ High density map –51 markers + Resistance phenotype ¾ Map spans a distance of 4.45cM ¾ Averages 0.03cM between markers ¾ 34 markers placed < 1cM from resistance HighHigh InformationInformation ContentContent FingerprintingFingerprinting (HICF)(HICF) ¾ Each BAC screening provided more than one BAC hit ¾ High Information Content Fingerprinting (HICF) was carried out to merge similar BACs ¾ BACs were fingerprinted and assembled using the program FPC v9.3 at a threshold of 1 x e‐35 ¾ HICF assembled 22 contigs and 23 singletons

FPC output showing the BACs in a single contig PhysicalPhysical MappingMapping ofof EFBEFB resistanceresistance regionregion

¾ High resolution genetic map and HICF data allowed construction of a physical map. ¾ Sizes of the BACs were estimated using PFGE (Pulse Field Gel Electrophoresis) PhysicalPhysical MapMap ofof thethe EFBEFB resistanceresistance regionregion IdentificationIdentification ofof resistantresistant contigcontig

¾ A single recombination event was observed between W07‐375 and resistance ¾ A single recombination event was observed between resistance and HICF13 ¾ W07‐375 and HICF13 are from the same contig ¾ Size of the contig is ~ 150kb ¾ Resistant contig consists of 3 overlapping clones ƒ 87B7 1 Recombinant R 1 Recombinant Plant ID UY44 locu Plant ID VC55 ƒ 66C22 s ƒ 43F13 SequencingSequencing ofof WholeWhole BACsBACs

¾ BACs in contigs from 173‐500, AA12‐850, W07‐375 and X01‐825 ¾ Sequencing with Illumina IIx genome analyzer ¾ 9 BACs per lane were multiplexed using 3bp barcoded adapters ¾ 80bp paired end sequencing DataData analysisanalysis andand BioinformaticsBioinformatics

¾ 80bp paired‐end reads were sorted according to the adapter barcodes using perl scrip bcsort_pe.pl (brianknaus.com) ¾ De novo assembly of sequences carried out in three steps 1. Sequences assembled using Velvet de novo short read assembler 2. Sequences reassembled using SOPRA de novo assembler 3. Contigs from Velvet and SOPRA were aligned, trimmed and corrected using CodonCode Aligner software CodonCodeCodonCode AlignerAligner AssemblingAssembling ofof BACsBACs

ƒ Number of contigs : min 1 –max 13 per BAC ƒ Largest contig : 99kb ƒ Smallest contig:350bp ƒ Approximate coverage: min 60% ‐ max 100% AbAb initioinitio GeneGene AnnotationAnnotation

¾ BACs sequenced with Illumina IIx genome analyzer were searched for the potential genes ¾ Gene prediction carried out using the program AUGUSTUS ƒ Arabidopsis as the gene prediction model ƒ RNA‐Seq data from ‘Jefferson’ as the source for transcript support (Data from Mockler’s lab) ƒ Predicted genes with a cutoff of > 60% of transcript support from RNA‐seq data were analyzed ƒ Amino acid sequences of predicted genes were BLAST(P) searched for protein homology 1 4 2 6 5 7 3 GenesGenes predictedpredicted byby AugustusAugustus

Total 233 73 43 32 GeneGene productsproducts predictedpredicted inin aa BLASTPBLASTP searchsearch PotentialPotential CandidateCandidate GenesGenes inin EFBEFB resistanceresistance regionregion Predicted gene Contig4_g19 ¾ Belongs to p‐loop NTPase superfamily ¾ Includes NBS‐LRR type disease resistance proteins ¾ NBS‐LRR forms the major class of R genes so far identified

Predicted gene Contig4_g25 ¾ Belongs to F‐box superfamily ¾ Plant F‐box genes –one of the largest multi gene superfamilies ¾ Control many important biological functions including disease resistance SummarySummary

¾ A high resolution genetic map with 1488 seedlings was constructed with 51 markers with an average distance of 0.03cM between markers ¾ A physical map of the EFB resistance region was constructed and the resistance gene was assigned to a single contig of three BACs ¾ Whole BACs in the resistance region were sequenced with Illumina IIx Genome analyzer ¾ Ab initio gene annotation of the sequences identified two potential resistance gene candidates: NBS‐LRR and F‐box genes